Microbiology Case Study: A 24 Year Old Male with Painful Pustules

Case History and Laboratory Findings

A 24 year old male presented to an urgent care clinic with the complaint of painful pustules on his scrotum for the past 4-5 days. He claims that he had not had any recent exposure to sexually transmitted diseases (STD) or to new sexual partners. A month prior to presentation, he was seen for STD evaluation and was treated empirically for suspected exposure.  Testing at that time was negative.  The patient was unsure if these pustules were exacerbated by his work, in which he is in tight, hot spaces doing manual labor. His review of systems was otherwise negative. A swab of one of the pustules was sent for gram stain and culture which showed the following:

neissgonopust1.png
Image 1. Gram stain from a swab illustrating small intracellular Gram negative diplococci (100x, oil immersion).
neissgonopust2
Image 2. Chocolate agar showing small to medium, flat, grey-brown, and moist colonies.

MALDI-TOF identified the organism to be Neisseria gonorrhoeae.

Discussion

N. gonorrhoeae is a gram negative diplococci and the second most common cause of sexually transmitted infections in the United States. The diplococci are described as having adjacent flattened sides giving it the appearance of the letter “D”. N. gonorrhoeae is classically described as a fastidious organism, requiring specialized media (Chocolate, modified Thayer-Martin, Martin-Lewis, New York City agar, etc.) and an enhanced CO2 environment in order to grow, though it is also known to grow on blood agar. Colonies are small to medium in size and are described as flat, grey-brown, and moist. Biochemically, N. gonorrhoeae is catalase and oxidase positive, and is a glucose fermenter.

Neisseria gonorrhoeae can infect the epithelium of the urethra, cervix, pharynx, rectum, and conjunctiva. Infection at these sites results in pain, irritation, and purulent discharge. Dissemination to other locations, such as the skin and joints, can also occur. Though dissemination is uncommon, associated symptoms include: skin sores, fever, migratory polyarthritis, tenosynovitis, and pauciarticular septic arthritis.  N. gonorrhoeae infections typically present as acute urethritis with associated discharge.  Infections are symptomatic in 10% of males and upwards of 70% of females, putting females at a higher risk of developing ascending infections, and potentially, pelvic inflammatory disease. Disseminated infections occur much less commonly, happening in 0.5% to 3% of all gonococcal infections. Therapy guidelines recommend treating uncomplicated infections with intramuscular ceftriaxone and oral azithromycin. Treatment of disseminated infections is variable depending on patient symptoms, but can include a combination of ceftriaxone/cefotaxime and azithromycin/doxycycline, with variable route of administration and length of treatment times. Susceptibility testing is limited to testing for beta-lactamase activity, though in cases of suspected resistance, CLSI guidelines are available for further testing.

References

  1. McCormack WM, Stumacher RJ, Johnson K, Donner A. Clinical spectrum of gonococcal infection in women. Lancet 1977; 1:1182.
  2. Workowski KA, Bolan GA, Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines, 2015. MMWR Recomm Rep 2015; 64:1.
  3. Centers for Disease Control and Prevention. Sexually Transmitted Disease Surveillance, 2015. Atlanta, GA: US Department of Health and Human Services; October 2016.

 

-Clayton LaValley, MD is a 2nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Associate Professor at the University of Vermont.

Microbiology Case Study: A 56 Year Old Man with Bacteremia

Case History

A 56 year old man with a history of hypertension, asthma and COPD, bladder stones, congenital bladder abnormality, and bladder exstrophy presented to the ED with fatigue, generalized weakness, poor appetite, fever, and nausea. He had intermittent right flank pain and generalized abdominal pain. The patient also reported he had passed bladder stones in the past year.

In the ED he had an elevated peripheral white blood cell count of 12.9 x103/μl with a differential of 81% neutrophils, 11% lymphocytes, and 6% monocytes. His creatinine was elevated at 5.4 mg/dl. CT imaging of the urogenital tract showed hydronephrosis with evidence of infection.

Blood and urine specimens were sent for culture. The urine culture grew 10-50,000 cfu/ml Aerococcus urinae. After 30 hours of incubation, the anaerobic blood culture was positive for small, coccoid-looking gram positive rods (Image 1). The following bacteria grew on solid media under anaerobic conditions after 48 hours of incubation (Image 2).

actscha1
Image 1. Gram stain showing small, coccoid-looking Gram-positive rods
actscha2
Image 2. Tiny colonies growing on non-selective anaerobic media after 48 hours.

Discussion

The organism growing in the blood culture was identified as Actinotignum schaalii. A. schaalii is a facultative aerobe that prefers enriched media and grows best in anaerobic or 5% CO2 environments. It is a non-hemolytic, non-motile, and non-spore forming Gram-positive rod. A. schaalii is negative for catalase, oxidase, CAMP factor, and nitrate reduction, but is hippurate positive. It is also negative for lactose, mannitol, raffinose, sorbitol, and trehalose acid production.

Actinotignum schaalii was formerly named Actinobaculum schaalii. The Actinotignum genus now consists of A. urinale, A. sanguinis, and A. schaalii. A. schaalii is part of urogenital microbiota in healthy adults and can cause urinary tract infections, especially as it thrives in the humid atmosphere of diapered children or elderly adults. Isolation of this organism is associated with urinary incontinence, bladder cancer, catherization, neurogenic bladder, renal failure, prostate cancer, and chronic renal failure. The presence of A. schaalii in the urogenital tract can progress to bacteremia and rarely it can also cause groin abscesses.

Actinotignum schaalii is an under recognized uropathogen due to its fastidious nature—it is slowly growing and prefers enriched media grown under anaerobic conditions. None of these conditions are met as part of a routine urine culture as urine cultures are often finaled in < 48 hours, they may not use enriched media, and they are incubated under aerobic rather than anaerobic conditions. Because of these factors, A. schaalii rarely grows in urine culture despite it being a known colonizer of the urogenital tract and occasional uropathogen. Instead it is most commonly recovered from blood cultures as a result of urosepsis. When colonies do grow from blood culture, they are very difficult to identify by biochemical methods. A. schaalii is often misidentified by biochemical panels as Arcanobacterium spp. or Gardnerella vaginalis (API Coryne system, bioMerieux), Actinomyces meyeri (Rapid ID32A system, bioMerieux), or Actinomyces israelii (Rapid ANA II system, Remel). In our lab Bruker Biotyper MALDI-TOF MS was able to identify A. schaalii with high confidence. Based on the most current literature, Vitek MS misidentifies A. schaalii as G. vaginalis or A. meyeri. This data is from 2015, so it’s possible the Vitek MS spectra database has been updated and now identifies A. schaalii.

A. schaalii is routinely susceptible to all β-lactams but requires an extended duration of antimicrobial treatment compared to other uropathogens. It is resistant to trimethoprim/sulfamethoxazole and quinolones, both of which are commonly prescribed for urinary tract infection. Therefore, patients with A. schaalii are at risk for recurrent urinary tract infections due to inappropriate or inadequate antibiotic treatment.

Our patient was prescribed ciprofloxacin for presumed urinary tract infection prior to his bacteremia. He was switched to ceftriaxone upon hospital admission. His bacteremia was cleared and the patient was discharged 5 days later on an oral β-lactam antibiotic. 

References

  1. Lotte, R., L. Lotte, and R. Ruimy. “Actinotignum schaalii (formerly Actinobaculum schaalii): a newly recognized pathogen—review of the literature.” Clinical Microbiology and Infection1 (2016): 28-36. Le
  2. Le Brun, Cécile, et al. “Urinary tract infection caused by Actinobaculum schaalii: a urosepsis pathogen that should not be underestimated.” JMM Case Reports2 (2015).
  3. Manual of Clinical Microbiology, 11th edition

 

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois.

Microbiology Case Study: A 62 Year Old Male with Productive Cough

Case History

62 year old male from Louisiana with a medical history of COPD presents with fever, productive cough, weight loss, and a red nodule on the left hand. Chest x-ray shows interstitial and lobar infiltrates. Patient reports no recent travel history. Mycobacterial culture of the sputum is positive for an organism. MALDI-ToF of the sputum culture confirms the result.

Lab Identification

mycokans1
Image 1. Kinyoun stained long bacilli with a banded, ladder like pattern isolated from sputum culture.
mycokans2
Image 2. Bright lemon yellow colonies growing on LJ slant after exposure to light on the right compared with unpigmented control colonies on the left.
mycokans3
Image 3. Bright yellow colonies growing on 7H11 media after exposure to light on the right compared with unpigmented control colonies on the left.

Mycobacterium kansasii grows on 7H11 media and Lowenstein-Jensen (LJ) slants. The colonies appear smooth or rough and unpigmented when isolated in the dark. Upon exposure to light, colonies turn bright lemon yellow due to enhanced b-carotene production, which makes M. kansasii a photochromogen. M. kansasii stains positive with acid-fast stains. On Kinyoun stain, it shows long bacilli with a banded, ladder like pattern. M. kansasii is positive for nitrate reduction, catalase, urease, and tween hydrolysis. It is negative for niacin and pyrazinamindase. The identification of M. kansasii is confirmed by MALDI or DNA probe of an isolate.

Discussion

Mycobacterium kansasii is a slow growing photochromogen, an organism that grows unpigmented colonies in the dark but produces a bright lemon yellow pigment upon exposed to light3. It is also an acid-fast positive long bacillus that causes TB-like chronic pneumonia, which is the second most common non-TB mycobacterial infection after MAC in the AIDS population. There are five genotypes of Mycobacterium kansasii. Genotypes I and II infect humans, with I being the most prevalent. Because environmental sources of M. kansasii are rarely identified, isolation of M. kansasii from a culture is never considered a contaminant.

M. kansasii is usually found in the tap water in cities endemic for the infection. In the U.S., it is most prevalent in the central and southern states including Louisiana, Illinois, Texas, and Florida1. Internationally, it is most prevalent in Israel, Korea, France, Japan, Portugal, with the highest incidence in England, Wales, and among South American gold miners.

The majority of patients with M. kansasii infection have an underlying pulmonary disease such as COPD, bronchiectasis, or TB infection3. The clinical manifestation of M. kansasii infection includes a primarily unilateral cavitary infiltrate in the lungs without pleural effusions. Patients are generally older compared with those infected with TB. They present with productive cough, weight loss, fever, night sweats, and dyspnea. Symptoms are usually less severe but more chronic compared with those of TB pneumonia. M. kansasii can also present as cutaneous lesions similar to sporothrichosis. Lesions include nodules, pustules, red plaques, and ulcers1.

M. kansasii infection is diagnosed by chest X-ray or chest CT, positive respiratory culture, and clinical exclusion of other diagnoses2. The criteria for a positive culture include either two consecutive positive sputum cultures, one positive culture from bronchoscopy specimens, or one positive culture with compatible clinical symptoms2. The treatment of M. kansasii infection depends on the resistance of the organism to rifampin. Rifampin-sensitive organisms are treated with at least three drugs including rifampin, ethambutol, isoniazid, and pyridoxine1. Rifampin-resistant organisms are treated with three drugs including clarithromycin or azithromycin, moxifloxacin, ethambutol, sulfamethoxazole, or streptomycin1. Due to drug interaction, rifampin is contra-indicated among HIV patients taking protease inhibitors and nonnucleoside reverse transcriptase inhibitors. Because rifampin increases the metabolism of these HIV medications, it can lead to HIV drug resistance among these patients.

 

References

  1. Akram SM, Bhimji SS. Mycobacterium Kansasii. StatPearls. 2018. https://www.ncbi.nlm.nih.gov/books/NBK430906/
  2. Johnston JC, Chiang L, Elwood K. Mycobacterium Microbiol Spectrum. 2017;5(1):TNMI7-0011-2016. doi:10.1128/microbiolspec.TNMI7-0011-2016.
  3. Meraz A, Raheem S. Pulmonary Mycobacterium Kansasii Infection –A Tale of a Right Upper Lobe Cavitary Lesion [abstract]. Journal of Hospital Medicine. 2015; 10 (suppl 2). https://www.shmabstracts.com/abstract/pulmonary-mycobacterium-kansasii-infection-a-tale-of-a-right-upper-lobe-cavitary-lesion/. Accessed March 12, 2018.

 

-Ting Chen, MD is a 1st year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Associate Professor at the University of Vermont.

Microbiology Case Study: A 50 Year Old Man with Altered Mental Status

Case Histor

A 50 year old man presented to the emergency room with altered mental status, fever, and hypotension. His medical history was significant for quadriplegia, chronic indwelling urinary catheter with frequent urinary tract infections, and diabetes mellitus. The patient was intubated and started on vasopressors for presumed septic shock. CT scan of the abdomen showed dilated loops of bowel with possible ileus and pneumoperitoneum around the sigmoid colon.

Laboratory Identification

  • Clostridium difficile Fecal PCR: Negative
  • Fecal Bacterial Pathogen PCR: Negative for Salmonella spp., Shigella or Enteroinvasive E. coli spp., Campylobacter spp., or Shiga toxin producing genes
  • Urine culture: 2 strains of Pseudomonas aeruginosa and Klebsiella pneumoniae

Blood culture: At 41 hours, two of four blood culture bottles became positive (one aerobic bottle in each set). Gram stain showed budding yeast with pseudohyphae (Image 1). Growth of white, smooth colonies with foot-like projections was observed on agar (Image 2). MALDI-TOF analysis confirmed identification of the organism as Candida albicans.

candalbi1
Image 1: Gram stain of blood culture bottle demonstrating yeast form (left) and pseudohyphae (right).
candalbi2
Image 2: Colony morphology on chocolate agar demonstrating smooth white colonies with surrounding “feet.”

Discussion

Candida spp. are the most common yeast cultured from clinical specimens. Candida albicans is characterized macroscopically by growth of smooth, white colonies with surrounding “feet” which represent projections of pseudohyphae. The pseudohyphae are distinguished microscopically from true hyphae by constriction of the cells where they meet (with true hyphae, the cell walls will remain parallel). When incubated at 37 C for 2 hours C. albicans will produce germ tubes (extensions from the yeast cell representing an attempt at forming true hyphae).

Candida spp. are normal flora found in the gastrointestinal tract, mucous membranes, and skin. Invasive candidiasis is typically an opportunistic infection with the patient’s own, endogenous flora (although nosocomial spread also occurs). Although it can be normal skin flora, the growth of Candida spp. from a blood culture should be presumed to be pathogenic; treatment should be initiated, and attempts made to identify the source. Typically, invasive candidiasis arises from one of three sources: 1) colonization and biofilm formation on an indwelling intravenous catheter 2) dissemination from a deep nidus of infection (often urinary tract) or 3) translocation from the gastrointestinal tract. Patients in intensive care, those who are immunocompromised, at extremes of age, those who have been on broad spectrum antibiotics, and those with GI tract perforation or anastamotic leaks post-operatively are at greatest risk for developing invasive candidiasis.

C. albicans has historically been the most common species of yeast causing invasive disease. However, non-albicans species (C. glabrata, C. parapsilosis, C. tropicalis and C. krusei) now cause almost 50% of invasive candidiasis. This changing epidemiology is relevant because the different species demonstrate different susceptibility profiles to antifungal agents, including azoles and echinocandins. C. auris is another recently emerging species that shows resistance to multiple antifungal agents, and has been described as causing outbreaks of healthcare-associated infections.

Although the patient’s chronic indwelling urinary catheter raised suspicion for a urinary source of the candidemia, the urine culture failed to support that theory. Given the findings of the CT abdomen in this patient, translocation from the GI tract is favored as the source of the candidemia. The patient’s history of treatment with antibiotics for repeated urinary tract infections, however, may have placed him at greater risk for developing invasive candidiasis.

-Alison Krywanczyk, MD is a 4th year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Associate Professor at the University of Vermont.

 

Microbiology Case Study: A 49 Year Old with HIV and CNS Lymphoma

Case History 

A 49 year old African American female was transferred from an outside hospital due to orbital cellulitis. Her past medical history was significant for HIV, CNS lymphoma, for which she was taking methotrexate & rituximab, and type II diabetes. Her vitals were: blood pressure 181/145, heart rate 145, temperature 98.6°F and respiratory rate 20. On physical examination, her right eye was bulging, with conjunctiva & eyelid swelling, and her iris was non-reactive. Scant serous drainage was noted. Admission labs showed a normal white blood cell count (9.8 TH/cm2), glucose of 211 mg/dL (normal: 74-106 mg/dL), hemoglobin A1C of 7.7% (normal: 4.2-6.0%) and platelets were low at 41,000 TH/cm2. An infection was suspected and the patient was started on vancomycin and piperacillin-tazobactam. She had a head CT scan which showed right periorbital cellulitis and diffuse sinus disease but no abscess formation. Nasal endoscopy was performed and extensive adhesions & black colored, necrotic tissue of the right nasal cavity was noted in addition to whitish debris, consistent with fungal overgrowth extending into the nasopharynx. Biopsies were taken for frozen section and bacterial & fungal culture and Infectious Disease was consulted for management of a probable rhinocerebral fungal infection.

Laboratory Identification

rhiz1
Image 1. Biopsy of the right nasal wall showed tissue invasion and necrosis with broad, ribbon like hyphae that were pauciseptate and branched at right angles (H&E, 40x).
rhiz2
Image 2. Fluffy, white fungal growth on Sabouraud Dextrose and Sabouraud Dextrose with Chloramphenicol agars at 72 hours of incubation at 25°C. There was no growth on the Mycobiotic agar slant.
rhiz3
Image 3. Tape prep showed a round sporangium containing small sporgangiospores located directly below the rhizoids of the mold which is consistent with the diagnosis of Rhizopus spp. (lactophenol cotton blue, 40x).

Discussion 

Rhizopus spp. belong to the order Mucorales, are ubiquitous in the environment and are the most common etiologic agents of mucormycosis. Rhizopus spp. typically cause invasive infections in the nasal sinus, brain, eye and lung, particularly in patients that have uncontrolled diabetes, HIV or are immunosuppressed. Mucorales are angioinvasive, exhibit perineural invasion and there is usually thrombosis, infraction and necrosis of surrounding tissue. As the illness can progress quite rapidly, prompt diagnosis and treatment is necessary.

If a Mucorales is suspected, tissue specimens obtained during a surgical procedure should be sent for frozen section, direct examination with calcofluor white/KOH and fungal culture. On histologic exam or microscopic exam in the microbiology laboratory, the hyphae of Rhizopus spp. are wide & ribbion-like with few to no septations (pauci- or aseptate) and wide angle branching (90°) (Image 1). Further classification requires culture.

If a Mucorales is suspected, the tissue submitted for fungal culture should be minced into small pieces and directly applied to the appropriate fungal media. Grinding of tissue will kill the hyphae and result in no growth from culture. Mucorales will not grow on media containing cycloheximide. Rhizopus spp. grow rapidly within 1-4 days and start as white, fluffy colonies that become grey or brown in color as they mature (Image 2). The Mucorales are described as “lid lifters” due to their rapid growth and “cotton candy” like colonies that fill the plate. On lactophenol cotton blue prep, Rhizopus spp. have unbranching sporangiophores that terminate in a round sporangium and arise directly under well-developed rhizoids (Image 3). The sporangium ruptures when mature and releases many oval sporangiospores.

Treatment of patients with mucormycosis is usually a dual approach with wide surgical excision and amphotericin B, which has been shown to be an effective anti-fungal drug in the majority of Mucorales. In contrast, voriconazole has poor activity against these isolates. If susceptibility testing is needed, CLSI provides reference broth microdilution guidelines. In the case of our patient, due to the grave prognosis of her condition, in addition to her other comorbidities, the family elected for comfort care measures only and board spectrum anti-fungals were not started.

 

Stempak

-Lisa Stempak, MD, is an Assistant Professor of Pathology at the University of Mississippi Medical Center in Jackson, MS. She is certified by the American Board of Pathology in Anatomic and Clinical Pathology as well as Medical Microbiology. She is the Director of Clinical Pathology as well as the Microbiology and Serology Laboratories.  Her interests include infectious disease histology, process and quality improvement and resident education.

Microbiology Case Study: A 22 Year Old Female with Wound Infection

 

Case History

22 year old female with a past medical history of scoliosis presents for routine follow-up after hospital discharge for post-op wound infection following a spinal fusion surgery. Patient had an anterior and posterior spinal fusion with allograft and hardware on 1/18/18. She had a laminectomy and irrigation for post-op epidural hematoma on 1/19/18. Subsequently, she developed a lumbar spine abscess and underwent irrigation and debridement of the abscess on 3/1/18. Two operative cultures of the left paraspinal musculature grew only tiny clear colonies on the anaerobic blood plates. Gram stain of these colonies did not show any organism. MALDI-ToF MS identified these colonies as Mycoplasma hominis which was confirmed at a reference laboratory by PCR. The patient was given daptomycin plus levofloxacin. Since discharge from the hospital, she had wound healing with intermittent discharge.

Lab Identification

Mycoplasma hominis requires a specific rich and complex agar medium for growth and grows tiny colonies on standard media such as Columbia agar. In a patient with urogenital disease, Mycoplasma hominis is diagnosed with a urogenital specimen culture and confirmed by PCR. In a patient with spinal hardware infection, Mycoplasma hominis is diagnosed by a culture of infected tissue with PCR confirmation.

Discussion

Mycoplasma is a bacteria that lacks a cell wall and contains the smallest bacterial genome totally sequenced. Due to its lack of cell wall, Mycoplasma cannot be visualized with a Gram stain, and it is innately resistant to b-lactams.1 Due to its small bacterial genome, 580 kpb, it cannot be detected by light microscopy and requires complex nutrients for growth1.

Mycoplasmas are frequently part of the oropharyngeal and genital tract flora among healthy subjects.1 There are more than 200 Mycoplasma species, of which 13 have been isolated from humans. Only 6 species, among which 5 are pathogens, live in the urogenital tract.2 As one of the Mycoplasma species detected in the genitourinary tract, M. hominis can be either a pathogen or part of the normal flora.1 Colonization with M. hominis is associated with younger age, lower socioeconomic status, multiple sexual partners, African American ethnicity, and hormonal status.1 Infection with M. hominis is more common among pregnant women.1

Mycoplasma hominis is associated with genital infections in females but not in males. Examples of infections include pelvic inflammatory disease and bacterial vaginosis.1 In addition, it is responsible for pregnancy-related infections such as chorioamnionitis and post-partum fever secondary to endometritis.1 Moreover, M. hominis is associated with infections of the newborns, meningitis among premature babies, and low birth weight among neonates.1 Lastly, M. hominis can lead to extragenital infections including spinal hardware infections, septic arthritis, retroperitoneal abscess, hematoma infection, and osteitis.1

Infections by Mycoplasma hominis are infrequent and difficult to confirm prior to the start of empiric therapy.2 Urogenital and systemic infections due to Mycoplasma hominis are treated with oral tetracycline.1 For organisms resistant to tetracycline, fluoroquinolones are recommended.1 For wound infections or abscesses, doxycycline, clindamycin, or fluoroquinolones are recommended for at least 2 weeks.1 Drainage and debridement may be necessary.1

References

  1. Pereyre S. et Mycoplasma hominis, M. genitalium and Ureaplasma spp.  Antimicrobe http://www.antimicrobe.org/m06.asp
  1. Baum S. Mycoplasma hominis and ureaplasma urealyticum infections. (2017, Dec. 7th).  Last retrieved on March 27, 2018 from https://www.uptodate.com/contents/mycoplasma-hominis-and-ureaplasma-urealyticum-infections

 

-Ting Chen, MD is a 1st year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Associate Professor at the University of Vermont.

Microbiology Case Study: A 70 Year Old Female with Bronchiectasis

Case History 

A 70 year old female presents with bronchiectasis with acute exacerbation. She is a non-smoker, although claims to have been exposed to secondhand smoke, and she has chronic sinusitis. The patient recently traveled to Savannah, Georgia where she developed a productive cough. She was prescribed doxycycline and was then sent home. She returned to the pulmonary clinic for a follow up consultation after her cough worsened.

Laboratory Identification

hflu1
Image 1. Intracellular gram negative coccobacilli with polymorphonuclear cells found in the sputum smear (100x oil immersion).
hflu2
Image 2. The predominant organism found in this patient’s sputum culture is growing 4+ on chocolate agar, but not growing on blood and MacConkey agars.
hflu3
Image 3. Close up of chocolate agar showing 4+ growth of wet, translucent colonies.

The Gram stain and smear showed 4+ neutrophils, 4+ gram negative coccobacilli and little to no mixed respiratory flora. The following day, the culture grew 1+ respiratory flora on the blood plate, no growth on the MacConkey plate, and 4+ translucent colonies on the chocolate plate. 

Discussion 

The predominant organism was identified by the MALDI-TOF as Haemophilus influenzae. The Gram stain and culture findings are consistent with the MALDI-TOF identification. H. influenzae is an oxidase positive, gram negative coccobacilli known for its requirement of X (hemin) and V (NAD) factors found in chocolate agar. Because of its growth requirements, H. influenzae will not grow on MacConkey agar despite being a gram negative organism. It may be cultured on blood agar if the agar is inoculated with an organism such as Staphylococcus aureus, which can provide the V factor, while the X factor is provided by the agar itself. This phenomenon is known as satelliting. Identification of H. influenzae may also be done using a Haemophilus ID Quad plate. Each section of the plate contains varying factors and allows for Haemophilus identification to the species level based on the growth and hemolysis pattern.

H. influenzae is normal flora of mucous membranes and frequently colonizes the human oral cavity and upper respiratory tract. Commonly, H. influenzae causes pneumonia, as with our patient, bronchitis, and ear infections. However, it is also a known cause for bacterial meningitis, endocarditis, and osteomyelitis. Transmission of H. influenzae occurs through respiratory droplets so proper PPE precautions must be taken by clinicians when working with infected patients. It is important for laboratory professionals to work with the organism using proper PPE and BSL-2 practices and plating of respiratory specimens should occur in a biosafety cabinet.

Susceptibility testing is not routinely performed on isolates of H. influenzae. β-lactamase production can be determined by using nitrocefin, a chromogenic cephalosporin spot test. 

References

  1. Haemophilus influenzae Disease (Including Hib). (2018, February 13). Retrieved June 28, 2018, from https://www.cdc.gov/hi-disease/index.html
  2. (2012, March 15). Retrieved June 28, 2018, from https://www.cdc.gov/meningitis/lab-manual/chpt09-id-characterization-hi.html. Identification and Characterization of Haemophilus influenzae
  3. Manual of Clinical Microbiology, 11th edition

 

MS

-Madaine Saguinsin, MLS (ASCP), graduated from Purdue University with a BS in Medical Laboratory Sciences and is a medical technologist at NorthShore University Health System. Her interests are microbiology and parasitology.

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois.