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Uncover interesting and unusual findings in the microbiology laboratory by browsing case studies, shared by your clinical and public health microbiology colleagues. Cases can be used as a teaching tool or to further your individual knowledge of the field.

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m1-1 . What is the MOST LIKELY diagnosis?

m1-2. The Gram stain from a skin pustule showed gram positive cocci in clusters. The organism grew on sheep blood agar (SBA) and was catalase positive. What is the most likely microorganism?

A. Staphylococcus aureus B. Staphylococcus epidermidis C. Streptococcus pyogenes D. Streptococcus pneumonia

Micro Case 2 (Path Slide 18) [ImageScope] [WebScope]

Clinical History: This 29-year-old male's illness began 10 weeks prior to death, with an episode of "flu". Two weeks later his urine became "smoky". He was found to have hematuria, albuminuria and elevated BUN (180 mg/dl). He died from a pulmonary embolus.

Image Gallery:

m2-1. What is the BEST diagnosis at the time of death?

m2-2. The throat culture obtained exhibited gram positive cocci in chains. It also showed beta-hemolysis on sheep blood agar (SBA) and was catalase negative. What was the most likely organism?

• Streptococcus pyogenes
• Streptococcus viridans
• Staphylococcus aureus

m2-3. An elderly wheelchair bound man had a history of recurrent urinary tract infections. He presents with new onset of fever, chills, and confusion. Blood cultures were drawn. The organism grown was non-hemolytic, catalase negative, and PYR positive. Gram stain showed gram positive cocci. What is the most likely organism?

• Staphylococcus epidermidis
• Staphylococcus saprophyticus
• Enterococcus species

m2-4. An elderly wheelchair bound man had a history of recurrent urinary tract infections. He presents with new onset of fever, chills, and confusion. Blood cultures were drawn. The organism grown was non-hemolytic, catalase negative, and PYR positive. Gram stain showed gram positive cocci. What is the MOST LIKELY organism?

Micro Case 3 (Path Slide 51) [ImageScope] [WebScope]

Clinical History: A 45-year-old male became ill approximately 2 to 3 weeks ago following an alcoholic spree. He had nausea, vomiting, dehydration, confusion and high fever. He died suddenly shortly after admission.

What is the MOST LIKELY diagnosis AND the likely causative agent?

m3-1. These images depict pneumonia in the stage of:

• Red hepatization
• Gray hepatization
• Abscess formation

m3-2. Community acquired atypical pneumonia can be caused by which of the following organisms?

• Legionella pneumophilia
• Klebsiella pneumoniae
• Mycoplasma pneumoniae
• Pseudomonas aeruginosa

Micro Case 4 (Path Slide 195) [ImageScope] [WebScope]

Clinical History: A 67-year-old male had rheumatic heart disease for thirty years. Three months prior to death he began to have episodes of fever and chills accompanied by signs of worsening congestive heart failure. Splinter hemorrhages and purpuric skin rashes were noted three weeks before death.

m4-1. What is the MOST LIKELY diagnosis?

m4-2. Most cases that present with these findings are caused by:

• Rickettsiae

m4-3. In this particular patient the MOST LIKELY causative organism was:

• Group A Streptococci
• Viridans Streptococci

m4-4. The organism MOST LIKELY to infect normal heart valves is:

• Group B Streptococci

m4-5. The organism MOST OFTEN associated with infective endocarditis in IV drug abusers is:

m4-6. The heart valve MOST OFTEN affected by infective endocarditis in IV drug abusers is the:

• Aortic valve
• Mitral valve
• Pulmonic valve
• Tricuspid valve

m4-7. Which of these organisms are normal flora of the throat and are associated with dental caries, brain abscesses, and endocarditis?

• Streptococcus pneumoniae
• Peptostreptococcus species

ANSWER  

m4-8. A patient with suspected infectious endocarditis has a St. Jude’s prosthetic aortic valve and a fever of 38.6°C (101.5°F). Blood culture shows non-hemolytic, small, white colonies. The organism was Gram positive, catalase positive and coagulase negative. What is the MOST LIKELY organism?

• Stapylococcus epidermidis

m4-9. A throat culture grows normal oropharyngeal flora. What alpha-hemolytic organism is most likely to be isolated on the blood agar?

A. Staphylococcus epidermidis B. Staphylococcus viridans C. Streptococcus pneumoniae D. Peptostreptococcus species

m4-10. A throat culture grows normal oropharyngeal flora. The coagulase test for the organism is positive. What is the genus and species of the organism?

A. Staphylococcus aureus B. Streptococcus agalactiae C. Streptococcus pyogenes D. Staphylococcus epidermidis

Micro Case 5

Clinical history: A 33-year-old female dairy farmer develops a severe headache and neck stiffness. On physical examination, her temperature is 38.2°C.  She has no papilledema.  A lumbar puncture is performed, and a Gram stain of the CSF obtained shows many short, gram-positive rods.

m5-1. Based on the clinical findings presented, what is the most likely causative agent in the case above?

m5-2. A sputum gram stain of an elderly person with cough and fever shows gram positive cocci in pairs. What is the most likely organism?

• Haemophilus influenzae
• Enterobacter species

m5-3. A 10-month-old child of a family from Mexico living in Durham was noted by his mother to have a grand mal seizure with shaking of arms and legs by the description given to the EMT. The EMTs found the child limp and unresponsive. In the emergency room the child’s fever was 39.5°C (103.1°F). Blood cultures and lumbar puncture were performed. CSF findings were as follows:

• cell count of 4000
• glucose 20mg/dl
• protein 125mg/dl.

Gram stain showed PMNs and occasional Gram-negative coccobacillary organisms. The organism grew on chocolate agar but not on sheep blood agar or MacConkey’s agar. What is the most likely organism in this case?

• Neisseria meningiditis
• Moraxella catarrhalis
• Streptococcus pneumonia

Micro Case 6 (Path Slide 123) [ImageScope] [WebScope]

Clinical History: A 25 year-old woman had pelvic pain, fever, and vaginal discharge for 3 weeks. On physical examination, she has lower abdominal adnexal tenderness and a painful, swollen left knee.  Laboratory studies show WBC count of 11,875/mm 3 with 68% segmented neutrophils, 8% bands, 18% lymphocytes, and 6% monocytes.

m6-1. What is the MOST LIKELY diagnosis AND the likely causative agent?

m6-2. What is a likely complication of this disease?

• Ectopic pregnancy
• Infertility
• Tubo-ovarian abscess
• ALL of the above

m6-3. What organisms are most likely to cause this disease?

m6-4. Which of the following statement about this disease is FALSE ?

• It is usually associated with endometriosis
• May be complicated by strictures and infertility
• May also involve the adjacent ovary
• May result in a hydrosalpinx
• May be complicated by septicemia
• ALL of the above statements are false regarding this disease.
• NONE of the above statements are false regarding this disease.

m6-5. The following organisms are commonly responsible for pelvic inflammatory disease EXCEPT :

• Aspergillus
• Streptococcus
• Staphylococcus

m6-6. A 25-year-old male presents with urethritis. The Gram stain shows intracellular gram positive cocci. What is the most likely organism?

• Chlamydia trachomatis
• Ureaplasma species
• Neisseria gonorrhoeae
• Haemophilus ducreyi

Micro Case 7 (Path Slide 9) [ImageScope] [WebScope]

Clinical History: A 51-year-old male had a " neurogenic bladder ", caused by a spinal cord tumor. He had multiple bladder infections which were treated with antibiotics. He had surgery to remove the tumor. Postoperatively, he developed fever and costovertebral angle tenderness which did not respond to antibiotics. He expired and an autopsy was performed.

m7-1. What is the BEST diagnosis?

• Acute pyelonephritis
• Acute glomerulonephritis
• Chronic pyelonephritis
• Chronic glomerulonephritis

m7-2 . ALL of the following are risk factors for this condition EXCEPT :

• Reflux nephropathy
• Congenital vesicoureteral reflux
• Posterior urethral valves
• Acetaminophen (Paracetamol) overdose
• Urolithiasis

m7-3 . A urine culture from an 18-year-old woman with similar symptoms also grew a pure culture of more than 100,000 colonies/ml of an organism on sheep blood agar (SBA) and MacConkey’s agar. The gram stain also showed gram negative rods. What is the most likely organism?

• Escherichia coli

Micro Case 8 ( Pathology Slide 39) [ImageScope] [WebScope]

Clinical History: 58-year-old African American female had been hemiplegic on the right side for 3 months prior to death. She developed malaise, fever and chills after visiting with her grandchildren. Her infection progressed. She developed dyspnea and expired.

Image Gallery

m8-1. Based on these clinical findings, what is the best diagnosis and likely causative agent ?

m8-2. What is the most likely cause of this disease in most adults?

M8-2. What organism would be most likely to cause a persistent infection in cystic fibrosis?

Micro Case 9

Clinical history: A 52-year-old homeless, alcoholic man had a fever and a cough productive of thick sputum that worsened over several days. His temperature is 38.2°C.   Diffuse crackles are heard at the right lung base. Laboratory studies are as follows:

• hemoglobin: 13.3 g/dL
• hematocrit: 40%
• platelet count: 291,8000/mm 3
• WBC count: 13,240/mm 3 with 71 segmented neutrophils, 7% bands, 16% lymphocytes, and 6% monocytes.

m9-1. Based on these clinical findings, what is the likely causative agent?

m9-2. An 18-year-old Duke freshman presented to student health with severe headache, fever, and disorientation. A lumbar tap was performed with the following results:

• cell count: 300 with 100% PMN
• glucose: 10 mg/dl
• protein: 100 mg/dl.

The gram stain of the spinal fluid revealed numerous PMNs with intracellular gram-negative diplococci. What is the most likely organism?

• H aemophilus influenza
• Neisseria meningitides

Micro Case 10

Clinical history: A 66-year-old man incurs extensive thermal burns to his skin and undergoes skin grafting procedures in the surgical intensive care unit.  Two weeks later, he has increasing respiratory distress.  Laboratory studies show hemoglobin of 13.1 g/dL, hematocrit 39.2%, platelet count 222,200/mm 3 , and WBC count 4520/mm 3 with 15% monocytes. A chest radiograph shows extensive bilateral infiltrates with patchy areas of consolidation. Image Gallery:

m10-1. Based on these clinical findings, what is the likely causative agent?

m10-2. A sputum specimen from a cystic fibrosis patient grew Gram negative rods on sheep blood agar and MacConkey’s agar. The organism was oxidase positive. What is the most likely organism?

Micro Case 11

Clinical history: A suspicious envelope arrived for sorting at rural post office. The envelope was opened and found to contain white powder. Approximately two days later, the postal worker who handled the letter developed cutaneous boils, which were and 1 to 5 cm in diameter with central necrosis and eschars. He and his wife also developed a mild nonproductive cough with fatigue, myalgia for 72 hours, followed by severe dyspnea, diaphoresis and cyanosis.  Temperature of 39.5°C, pulse 105/min, respiration 25/min, and blood pressure 85/45mm Hg.  Crackles were heard at the lung bases. A chest xray shows a widened mediastinum and small pleural effusions. WBC count of 13,130/mm 3 , hemoglobin 13.7g/dL, hematocrit 41.2%, MCV 91 um 3 , and platelet count 244,000/mm 3 . Both died despite antibiotic therapy. Several cattle, horses, and sheep on the postal worker's farm also died.

m11-2. In a somewhat related case, when Pharaoh did not heed Moses to the let the captive Hebrews go, a series of plagues fell upon the land of Egypt. In the fifth plague, large domesticated mammals including cattle, horses, and sheep died. This was followed by a plague in which the Egyptians developed cutaneous boils. Some developed a mild nonproductive cough associated with fatigue, myalgia, and low grade fever over 72 hours, followed by a rapid onset of severe dyspnea with diaphoresis and cyanosis. Despite antibiotic therapy with both ciprofloxacin and doxycycline (had they been available), many of those affected would die. Which of the following organisms is most likely to have produced these findings?

• Bacillus anthracis
• Herpes simplex virus
• Mycobacterium leprae
• Yersinia pestis

Micro Case 12 (UMich Slide 017) [ImageScope] [WebScope]

Clinical history : A 45-year-old woman is being treated in the hospital for pneumonia complicated by septicemia. She has required multiple antibiotics and was intubated and mechanically ventilated earlier in the course. On day 20 of hospitalization, she has abdominal distention. Bowel sounds are absent, and abdominal radiograph shows dilated loops of small bowel suggestive of ileus. She has a low volume of bloody stool.

m12-1. Based on these clinical findings, what is the likely causative agent?

m12-2. Which of the following are appropriate specimen samples for anaerobe culturing:

• blood, spinal fluid, abscess aspirate
• deep tissue biopsy, sputum, blood
• cerebrospinal fluid, tissue and debridement from decubitus ulcer, bile

Micro Case 13

Clinical history: A 25-year-old man is involved in an accident in which he is ejected from the vehicle. He sustains a compound fracture of the left humerus and undergoes open reduction with internal fixation of the humeral fracture.  Several days later, he has marked swelling of the left arm and crepitus . 

m13-1. In a similar case, a middle aged woman with type 2 diabetes presents to the emergency room with a very painful right lower leg. She has a wound on that extremity and stated that she stumbled on a fallen tree limb in her yard. You observe that her lower leg is discolored and swollen with several areas of crepitus . The abscess fluid was sent to the microbiology laboratory for aerobic and anaerobic culture. The organism grew best on anaerobic cultures and was lecithinase positive. Gram stain shows gram positive rods and the organism show anaerobic growth on egg yolk agar. What is the most likely organism?

• Fusobacterium nucleatum
• Bacteroides fragilis
• Clostridium perfringens
• Peptostreptococcus spp.

Micro Case 14 (Path Slide 451) [ImageScope] [WebScope]

Clinical History: A 4-year-old female had a gradual onset of fever, productive cough, anorexia and diarrhea about eleven days prior to death. The breath sounds were harsh, and a few cracking rales were heard over the right base posteriorly.

m14-1. Based on these clinical findings, what is the likely causative agent?

m14-1. Which of the following is the BEST diagnosis?

• Ghon complex
• Miliary (disseminated) tuberculosis
• Foreign body reaction to talcum powder
• Atypical pneumonia
• H1N1 influenza

Micro Case 15

Clinical history: A 35-year-old man with HIV complains that he has had a "bad" taste in his mouth and discoloration of his tongue for the past 6 weeks.

m15-1. What is the MOST LIKELY diagnosis?

m15-2. In a separate case, a blood culture from a neutropenic (<100 neutrophils/ul) 50-year-old woman on broad spectrum antibiotics grew a yeast. Tests for germ tubes were positive in the microbiology laboratory. What is the likely organism?

• Candida albicans
• Candida tropicalis
• Candida pseudotropicalis
• Candida parapsilosis

Micro Case 16

Clinical history: A 44-year-old diabetic woman developed facial pain over the past 24 hours.  She has become lethargic and obtunded. There is swelling with marked tenderness over the left and right maxilla, bilateral exophthalmos, diffuse abdominal pain, poor skin turgor, and dry mucous membranes. Her temperature is 37.7°C. She has tachycardia, but no murmurs, and tachypnea; the lung fields are clear.

m16-1. What is the MOST LIKELY diagnosis and the best treatment option?

m16-2. In a separate case, A 22-year-old with non-Hodgkins lymphoma was profoundly neutropenic after induction chemotherapy and developed fevers. Broad spectrum IV antibiotic therapy was administered, but fevers continued. Chest x-ray showed new bilaterial fluffy pulmonary infiltrates. A bronchoscopy was performed which showed hyaline, septate hyphae with acute-angle branching. What is the MOST LIKELY organism?

• Blastomyces dermatitidis
• Candida species
• Aspergillus spp

Micro Case 17

Clinical history: A 50-year-old resident of Phoenix, Arizona, has a cough that has persisted for 1 month.  On physical examination, his temperature is 38.1°C. A chest radiograph shows 3.5-cm opacity with central cavitation in the right apical region.  An open lung biopsy is performed to exclude cancer.

m17-1. Which of the following organisms is MOST LIKELY to be responsible for these findings?

• Aspergillus fumigates
• Coccidioides immitis
• Histoplasma capsulatum
• Mycobacterium tuberculosis

Micro Case 18

Clinical history: For the past 3 weeks, a 52-year-old man has had a chronic cough with a low-grade fever. On physical examination, his temperature is 37.4°C.  A chest radiograph shows bilateral, scattered, 0.3- to 2-cm nodules in the upper lobes and hilar adenopathy.  A fine needle aspirate of one of the nodules shows inflammation with mononuclear cells, including macrophages that, with PAS or silver stains, show intracellular, 2- to 5-um, rounded, yeast-like organisms.

m18-1. Which of the following infectious diseases is MOST LIKELY to produce these findings?

• Coccidioidomycosis
• Candidiasis
• Cryptococcosis
• Histoplasmosis
• Blastomycosis

Micro Case 19

Clinical history: For the past month, a 68-year-old patient has had painful oral abcesses, fever, and a cough productive of yellow sputum.  On physical examination, there is dullness to percussion at the left lung base. A chest radiograph shows areas of consolidation in the left lower lobe.  Despite antibiotic therapy, the course of the disease is complicated by abscess formation, and he dies.

m19-1. Based on these clinical findings, what is the BEST diagnosis AND the likely causative agent?

m19-2. In a separate case, a middle aged man presented to his physician with a persistent cough of two months following an extended overseas trip to visit relatives. He had also noted a 10 pound weight loss and night sweats. A sputum was sent to the microbiology laboratory for routine bacterial culture and AFB culture. Kinyoun stain of his sputum was positive. What is the likely causative agent?

• Actinomyces spp
• Nocardia spp.

Micro Case 20

Clinical history: A 50-year-old man post lung transplant was admitted to hospital with fever, chills and cough. Chest x-ray showed multiple small abscesses within a right middle lobe infiltrate. The patient had been treated with prednisone and azathioprine daily for rejection. Gram stain of a bronchoalveolar lavage of the right middle lobe shows gram positive rods. Modified acid fast stain shows partially acid fast bacilli.

m20-1. What is the most likely organism?

m20-2. Nocardia species are:

• branching, gram negative bacilli
• partially acid fast, beaded gram positive filaments
• acid fast, branching gram negative filaments
• partially acid fast, gram variable bacilli

Micro Case 21

Clinical history: An HIV positive male presented in clinic with confusion and disorientation. He had a fever 38.5°C and photophobia. His CD4 count was 80/ul. A lumbar puncture was performed. It showed 32 WBC/ul with 89% lymphocytes, and 6% monocytes, glucose of 22mg/dl, and protein of 89mg/dl. Gram stain showed yeast and India ink showed a thick capsule.

m21-1. Which of the following is the most likely pathogen?

• Cryptococcus neoformans
• Hemophilus infulenzae
• West Nile virus

m21-2. In a separate case, A 35-year-old man who received kidney transplantation was being treated with cyclosporine, azathioprine, and high doses of corticosteroids.  While on this regimen, the patient began to experience headaches and became lethargic.  A clinical diagnosis of meningoencephalitis was made.  He died 7 days later.  Autopsy showed a gelatinous meningeal exudate, and on sectioning of the brain, multiple small cyst-like areas were seen.  Microscopic examination showed areas containing rounded structures with a prominent capsule that stained brightly with mucicarmine. 

What is the most likely organism?

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• v.16(2); 2015 Dec

Updated Cases for Medical Microbiology

Review of:   Cases in Medical Microbiology and Infectious Diseases , 4  th ed.; Peter H. Gilligan, Daniel S. Shapiro, and Melissa B. Miller; (2014). ASM Press, Washington, DC. 589 pages. 

The fourth edition of Cases in Medical Microbiology and Infectious Diseases is a well-organized compendium of real-life case studies focused on the practical applications of medical microbiology. Primarily written for medical students to help them study for board exams and infectious disease rotations, this volume may also be useful for instructors of upper-division undergraduate microbiology courses.

The fourth edition contains timely updates reflecting the emergence of many new infectious diseases and an increase in multi-drug-resistant infections. In addition, this new edition encompasses information about the latest molecular diagnostic tools such as MALDI-TOF, transcription-mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), and strand displacement amplification (SDA) assays and incorporates these into the cases. There is an entire chapter devoted to the explanation of these different diagnostic approaches. Each color-coded chapter of the book corresponds to a particular organ system and begins with an introduction to the incidence and transmission of diseases associated with that system. Important pathogens for each system are noted in a table at the beginning of each chapter, with information about the general characteristics of the pathogen, its reservoir, and disease manifestations. The main focus of each chapter is the pathogens’ characteristics, their associated diseases, clinical manifestations, and an appropriate course of treatment.

The purpose of the case studies is to foster critical thinking, problem-solving ability, and real-life diagnostic skills for future healthcare professionals. Each case is concisely summarized with the relevant presentation of symptoms, history, and clinical findings. Color photos and micrographs of clinical findings accompany each case to give the reader a “hands-on” feel for the experience of diagnosing a patient’s illness. Each case scenario description is followed by five to eight questions that prompt the reader to work through a diagnosis of the case. Notably, these questions are targeted toward areas of common confusion that could lead to misdiagnoses. Over half of the 74 cases presented in the fourth edition are new and those that appeared in the third edition have been updated. The fourth edition includes an additional “Advanced Cases” chapter providing exposure to many emerging infectious diseases and special cases of multidrug- resistant infections. An extensive “Case Discussion” is provided at the end of each scenario, with thorough, clearly-written explanations for each question. An up-to-date list of five to eight primary reference articles is also given at the end of each case. The last 50 pages of the book consist of a useful glossary of medical terms and an index that can be used to quickly find information on a particular topic.

Overall, the fourth edition provides a wealth of current resources for both students and instructors and can be used in a variety of ways in upper division microbiology courses (e.g., incorporating cases into lecture material, in-class group work, homework assignments).

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Case Studies: Microbiology

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Article Contents

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Cases in Medical Microbiology and Infectious Diseases, Fourth Edition

• Article contents
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• Supplementary Data

Lori Dockstader Racsa, Cases in Medical Microbiology and Infectious Diseases, Fourth Edition, Laboratory Medicine , Volume 46, Issue 1, Winter 2015, Page e18, https://doi.org/10.1309/LMHKL8HWSPZK1UE3

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The Fourth Edition of Cases in Medical Microbiology and Infectious Diseases is an updated case series that examines a broad range of clinical infectious diseases that will likely be encountered throughout a clinician’s career in medicine. It spans the knowledge base from the beginners’ level, including a glossary of basic medical terminology, through information widely held by those with more experience in general medical practice.

The case-based book has sections, divided by systems, varying from urogenital tract to central nervous systems. There is a final section focused on more advanced cases, including tropical medicine and zoonotic diseases. Each section starts with an overview about the disease processes and contains a table of the most common pathogens pertinent to the organ system, both of which are a useful study tool for students studying for board examinations.

Each case has multiple questions that ask readers to make a diagnosis based on clinical information; most include illustrated aspects of microscopic and microbiologic findings. The questions delve into different aspects of the disease and give a great overview of which microbiology laboratory tests may be applied to each situation. The book offers up-to-date information on molecular diagnostics that are now being used in most microbiology laboratories, replacing former standard culture techniques. The book also offers pharmacologic information on treatment for each disease, including susceptibility patterns and genes responsible for resistance patterns. In addition to diagnostics and treatment, most cases discuss the epidemiology of the given disease and give examples of outbreaks, when pertinent.

Cases in Medical Microbiology and Infectious Diseases is a great book for medical students and residents rotating through the infectious disease service. It offers a broad overview of the clinical aspect of each disease and diagnostics and treatment. The number of cases makes this book slightly longer than most medical students may want to read; however, the quality of information makes the text well worth the length. This book is an asset to medical technologists who want to learn more about the clinical disease process of organisms they are identifying, especially medical technology students. However, the book is not a bench top resource for the next steps in diagnostics in the laboratory. An overview of biochemicals and procedures used to identify organisms are presented—for instance, optochin, which is used for identifying Streptococcus pneumonia e. However, the zone of inhibition is undefined. Additional information with flowcharts and next steps in the decision making process would have made this book a bit more useful for technologists.

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Greenblum, S. Microbial adaptability in changing environments. Nat Rev Microbiol (2024). https://doi.org/10.1038/s41579-024-01046-w

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Published : 12 April 2024

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Microbiology Case Study: A 15 Year Old Male with Endocarditis

Case History

A 15 year old male with a past medical history significant for Tetralogy of Fallot (congenital heart defect), multiple valve replacements, chronic kidney disease, and prior Bartonella endocarditis. He presented with a “flu-like” illness including muscle aches, fevers, fatigue, and night sweats. His symptoms slowly dissipated after about three days. However, he had labs drawn including multiple blood culture sets which were all positive for growth.

Laboratory Findings

Gram stain showed gram positive bacilli and culture plates grew two morphologies of slow growing gray, granular and opaque colonies.This organism was identified by MALDI-TOF as Corynebacterium pseudodiphtheriticum.

The genus Corynebacterium comprises a collection of irregular-formed, rod-shaped or coccoid bacteria that are non-motile, catalase -positive, and non- spore -forming.

Corynebacterium pseudodiphtheriticum (previously designated as Corynebacterium hofmannii ) is a nonlipophilic, nonfermentive, urease- and nitrate-positive Corynebacterium species. 1 C. pseudodiphtheriticum is part of the usual oropharyngeal bacterial flora, including the nares and throat. It appears to play a role in preventing colonization of oropharyngeal epithelia by pathogenic bacteria.

Most commonly, C. pseduodiptheriticum is a pathogen of the respiratory tract with cases of nosocomial and community-acquired pneumonia, bronchitis, tracheitis, pharyngitis, and rhinosinusitis. Endocarditis is the second most common infection site, although very rare. Cases of urinary tract and wound infections have also been reported.

Treatment is usually with penicillin alone or in combination with aminoglycosides. Antibiotic susceptibility profiling of C. pseudodiphtheriticum isolates showed that resistance to oxacillin, erythromycin, clindamycin, and macrolides are common. 1

• Burkovski A. Corynebacterium pseudodiphtheriticum : Putative probiotic, opportunistic infector, emerging pathogen. Virulence . 2015;6(7):673–674. doi:10.1080/21505594.2015.1067747

-Nicole Mendelson, MD is a 1 st year Anatomic and Clinical Pathology resident at the University of Vermont Medical Center.

-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 .

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Fantastic case,……keep on All the best

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13: Putting it all Together—Case Studies in Microbiology

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In this lab, a series of stations will be set up around the room, each of which will present a microbiology case study. Some background information will be given to you, along with organisms on slides, growth media, etc. This is your opportunity to put together everything that you have learned about microbiology this semester. You will be expected to integrate the various pieces of information that are provided to answer some questions about the situation and come up with a diagnosis. The case studies will not be graded, and you will have an opportunity to work together to solve these problems. All of these activities will be good practice for your lab practical—try to enjoy the process!

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Clinical Case Study

You are a pathologist working in the lab at Daigger Memorial Hospital (located in central Oregon). The emergency room is sending you cultures collected from patients. Your job is to determine:

• The pathogenic bacteria causing the disease
• What disease is being presented
• What is the best treatment given the patient’s unique symptoms and history

Review the patient assessment form provided. Consider the type of sample taken and determine the relevant primary and secondary symptoms. Generally, samples taken in a clinical setting contain multiple organisms. In this case study, the sample will contain one contaminant organism and one causative agent.

Note: Watch for allergies. These may affect the appropriate treatment you will prescribe for the patient.

For some diseases, an irregularity in pulse or respiration, for example, may be indicative of the seriousness of the illness or may even be a direct indicator of the disease presented. Therefore, it is important that you understand what is considered “Within Normal Limits” for all assessment areas on the patient assessment form. Familiarize yourself with the acceptable normal ranges for pulse, respiration, oxygen saturation, blood pressure, and temperature for the age group your patient falls within.

Clinical Case Study Flowchart »

Patient Assessment Form »

Normal Flora According to Body System

Adapted from: Strohl, W.A. et.al. Lippincott's Illustrated Reviews: Microbiology. Lippincott Williams & Wilkins. Baltimore, MD. 2001 and Forbes, B.A. et.al. Bailey and Scott's Diagnostic Microbiology. Eleventh Edition. Mosby. St. Louis, MO. 2002

Pathogenic Organisms According to Body System

Adapted from: Gilligan, P.H. et.al. Cases in Medical Microbiology and Infectious Diseases. 2nd ed. American Society for Microbiology, Washington, D.C. 1997. Murray, Patrick R. Editor. Manual of Clinical Microbiology. 8th ed. ASM Press. Washington, D.C. 2003. Strohl, W.A. et.al. Lippincott's Illustrated Reviews: Microbiology. Lippincott Williams & Wilkins. Baltimore, MD. 2001

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Tracking and tracing to the root cause: case studies in microbial contamination

Posted: 11 April 2022 | Tim Sandle (Bio Products Laboratory) | 2 comments

In this article, pharmaceutical microbiologist and contamination control expert Tim Sandle presents three microbial contamination investigation case studies, highlighting the key lessons for pharmaceutical microbiologists to take away and the underlying importance of identifying the root cause of microbial data deviations.

Introduction

One of the main activities of the pharmaceutical microbiologist is with determining the contamination control strategy and proactively identifying measures to lower identified risks and, where risks remain of concern, to introduce monitoring. However, there will invariably be microbial recovery, from product (intermediate and finished), from water, cleanrooms and other utilities. A common term of these events is ‘microbial data deviations’, although other terminology can apply. 1 These microbial events require investigation and the importance of such an investigation is elevated where there is recurrence. While investigations are referred to in regulatory documents, there is a dearth of case studies to help guide microbiologists and Quality Assurance departments. This article presents three case studies. While the specific issues may or may not be of direct relevance, the areas examined and the thought processes will be of wider applicability.

Microbial investigations

Microbial data deviations can be categorised as:

• Out of limits results (OOL), which is often applied to environmental monitoring
• Out of specification results (OOS), which is typically used for pharmacopeia tests
• Out of trend (OOT), as defined in relation to a trend chart.

Microbial data deviations need to form part of the quality system and they require an investigation. The target time for microbiology investigations is to be completed within thirty days, against a procedure.

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The optimal way to gather evidence is to visit the area, where the role of the pharmaceutical microbiologist is mostly in the process area. 2 In addition, operators may need to be interviewed and the information should be captured and documented. A further important source of information can be the batch manufacturing or batch processing record. Visits and document reviews can be supported by additional sampling. With sampling, it is best practice to have a hypothesis developed in advance together with the numbers and types of samples detailed. More than one sampling session may be required to account for the variances with microbial distribution. 3 Further evidence should be gathered from accurate microbial species identification.

The three case studies draw on these essential elements and demonstrate how things can sometimes go wrong.

Case study 1: Microbial contamination in non-sterile manufacturing

The first scenario relates to tablet manufacture. The company in question noted repeated finished product failures with a Bacillus species, human skin commensals and Gram-negative rods. Individual tablets had a bioburden estimated to be more than 2,500 colony forming units (CFU), containing the mixed flora.

There was no obvious pattern in relation to the contamination, which was found throughout the batch. Further examinations indicated that multiple batches were found to be contaminated. The important starting point was to undertake microbial identifications to species level, as understanding the microorganisms can provide clues as to the habitat and possible origin. The pattern of different microorganisms also helps with comparing microorganisms isolated in the finished product with organisms recovered from other sources, such as water and the cleanroom, and to track contamination at different points in the process.

The main part of the investigation required a construction of a process flow chart, to enable the manufacturing process to be examined.

Figure 1 : General steps for tablet manufacturing.

The main manufacturing steps, together with initial observations, were:

• Wet mixing of raw materials – this is performed with purified water. The successful mixing of the powder is more difficult than mixing liquid and is based on achieving homogeneity. The ease of this is dependent upon the inherent cohesiveness and resistance to movement between the individual particles.
• Drying – with drying, it is important to keep the residual moisture low enough to prevent product deterioration and ensure free flowing properties. The drying process was found to leave the product as a cake with a high level of residual humidity within it.
• Bulking up – the bulking up process allowed multiple cakes to be transported to the tanks and added via a vacuum lance.
• Compression – compression was believed by the company to be antimicrobial. This was based on development work that provided evidence of a multi-log reduction in the microbial population.
• Packing – the packing process was into blisters, taking place within a controlled environment.

In addition, environmental monitoring data was examined. Here it was found that the routine monitoring of the manufacturing room had not recovered anything atypical. As well as the cleanroom, the purified water system had not seen any recent recoveries of microorganisms.

For the next stage of the investigation, the equipment in the manufacturing process was opened up and sampled microbiologically. In addition, testing was undertaken of the raw material, a substance of natural origin that was found to contain a low level of Gram-negative bacteria. It was of interest that these organisms were of the type seen in the failed batches, but the numbers recovered were relatively low. No organisms were recovered at the wet mixing stage.

The investigation proceeded to the drying stage, looking at the room equipment. One area examined, the extract pipework from the dryer, was found to have pools of condensate inside it. Due to a process improvement step, the extract was no longer being used due to the discontinuation of a product line. A previous version of the product used alcohol rather than water as the mixing agent (the extract was a safety precaution to remove the alcohol fumes). Additional testing revealed that the extract condensate was heavily contaminated with Gram-negative bacteria.

For the investigation of the bulking up stage, the operation of the lance was found to be satisfactory. However, increased environmental monitoring was undertaken and the active (volumetric) air samples taken within the lancing area cleanroom recovered a combination of Bacillus species and skin commensals. Other areas examined included:

• The tank – opened up via the main ‘lid’, further inspection was difficult because there was no manway
• The connection from tank wall to tank lid, which formed a lip about 10cm wide. The lip was covered in a old grey This residual powder was heavily contaminated with the microorganisms isolated from the finished tests.
• The lid of the tank, which had evidence of condensate on it.
• The room HEPA inputs – based on the walls (rather than being ceiling mounted, which would be more conventional), with cold filtered air continuously bathed the top of the tank.

With the study of the compression and packing lines, these recovered no microbial bioburden.

A further important element of any microbial investigation is with the trend. This is examined by constructing a trend chart and overlaying historical events. The trend analysis showed that the finished product failures, due to microbial contamination, began appearing after the alcohol containing product was discontinued.

The overall findings demonstrated a set of different failure modes:

• The wet cake enabled the survival of Gram-negative bacteria
• The lance activity pulled human skin and environmental isolates into the tank
• The old air trapped in the tank allowed condensate to form and
• the poor design of the tank retained the condensate, allowing it to hit the joining tank lip.
• The lip allowed contaminated powder to accumulate. The powder provided a nutritive source which facilitated bacteria to grow.

The reason that the issue appeared, and had not been noted previously, was because the discontinued alcohol containing product would have sanitised the filling equipment. In addition, the compression unit would have helped to have eliminated any low-level microorganisms that would have survived the alcohol step. The removal of the alcohol highlighted the poor controls through the process and led to conditions that enabled the proliferation of microorganisms.

Identifying these areas enabled appropriate corrective and preventative actions to be set. In particular, plant modifications and routine cleaning was introduced.

Enhancing rapid microbiology methods: how AI is shaping microbiology…

Case study 2: Environmental monitoring – the unexplained organism

The second case study, relates to a manufacturing operation within a Grade C cleanroom. From an environmental monitoring session, a working height surface contact plate of a conveyor belt recovered a high level of bacteria, with the plate being ‘too numerous to count’ (against an action level of 25 CFU/cm 2 ). As was customary, the isolates were Gram-stained and then a phenotypic identification was performed using a semi-automated commercial system. The predominant isolate was Yersinia pestis . Other isolates were a set of more typical skin biota of the Staphylococcus species.

The laboratory analyst did not question the Y. pestis identification and the importance of the organism was realised by the supervisor. This is a facultative anaerobic organism that can infect humans via the Oriental rat flea, resulting in plague. 4

The supervisor notified site management that ‘plague’ had been detected at the facility and unsurprisingly a state of panic ensued. Among the activities seriously being considered were site evacuation, notification of local authorities and placing everyone in the microbiology department under enforced isolation.

A more experienced microbiologist was called in and began assembling the facts. The microbiologist determined:

• The recovery of ‘bubonic plague’ from a conveyer belt did not make sense
• The environmental monitoring was performed on night shift
• Post incubation, the plate count was also performed on a night shift
• The microbial identification, following subculture, was also performed on night shift
• The supervisory review and subsequent alert of the discovery of plague was communicated on the night

The reason why the identification was most likely to be inaccurate was quickly established by:

• The apparent pestis was growing on the environmental monitoring agar, which was tryptone soya agar (TSA). In practice, pestis is isolated on stained smears of peripheral blood, lymph node specimens or from sputum. Y. pestis will grow as small, non- lactose fermenting colonies on MacConkey agar or Enterobacteriaceae selective agar, like EMB agar. These are supported by rapid microbiology assays. 5
• The particular colony did not look morphologically like a Gram-negative. Although there are limitations with visual identification, the colonial morphology of Gram-negative and Gram-positive bacteria can often be distinctive.
• A repeat microscopic investigation, requested by the experienced microbiologist, of the older original colony showed the presence of endospores, which pestis does not form.

The bacterium was not Y. pestis . The microbiologist established that the errors that led to the misidentification were:

• The original Gram stain was incorrect – a young colony was stained and the outcome was Gram-variable; however, the analyst selected Gram-negative as the outcome
• The incorrect Gram stain led to the selection of the incorrect identification biochemical test kit
• The bacteria still reacted or grew within a particular patter of biochemical wells, providing a pattern that that was closely matched to a database and the most probable result selected by the software. This was indicated to be pestis.

Rather than questioning the oddness of the result, an emotive response from the supervisor led to rapid escalation of the identification result and considerable business disruption.

From a human factors perspective, the night shift is not a popular timeslot for more experienced microbiologists to work, leading to less experienced personnel often being allocated. Furthermore, personnel are often tired and sometimes fatigue adds to the judgement process.

Case study 3: Sterile product out of specification

The third and final case study relates to a sterile active pharmaceutical ingredient (API) facility who manufacture an injectable dry powder. The product is sterile filtered into a pre-sterilised manufacturing line and then turned into a dry powder and offloaded via Grade A isolators.

With one batch, there was a subsequent sterility test failure. This led to the recovery of two different Bacillus species and an OOS investigation. During the first phase of the investigation, there was a second sterility test failure with the same type of product. This batch also recovered the same two Bacillus species, expanding the scope of the investigation.

The investigation was split into two parts: 6 the first looked at laboratory error and the second at the potential for a manufacturing failure (based on the typical first two phases of the OOS process). Across both parts, environmental monitoring was deployed to extensively sample manufacturing cleanroom air and surfaces; laboratory air and surfaces; isolator air and surfaces; and samples were taken from utilities like compressed air, nitrogen, water-for-injection (WFI), purified water, glycol, plant steam, heating ventilation and air conditioning (HVAC) ducting. A few environmental samples were also taken from support areas, such as the warehouses and controlled-non-classified (CNC) areas.

The environmental monitoring looked at the traditional techniques such as contacts and air samples. Some techniques had to be modified in order to allow for the sampling of air systems, glycol lines and high-pressure steam. The focus of the monitoring was less concerned with the numbers of organisms recovered and more with the types of microorganisms recovered, in an attempt to see if the two Bacillus species could be identified.

Many of the environmental monitoring samples recovered Bacillus in numerous places. The internal phenotypic identification system isolated the two specific species from the laboratory samples and one of the species from a production compressed air sample. The organisation concluded the results were a false positive based on finding the isolates in the laboratory, supported by the fact that two species had not been isolated together from the manufacturing plant. This was reported to senior management.

However, the isolates had also been sent away to an external laboratory for genotypic matching. The genotypic test results revealed that although the species of Bacillus were similar, the specific strains of the Bacillus organisms had not been found in the laboratory. In contrast, the species of Bacillus from the compressed air was genetically related and thus the same strain as the contaminant isolated from the two sterility test failures.

The dilemma was that the original phenotypic results had been reported with the point of origin directed to the laboratory, whereas the definitive data was now pointing towards a production related issue (the compressed air). The recommendation was made that the batches needed to be rejected, although the first reporting of the erroneous root cause caused considerable disagreement in senior management circles.

Lessons to be learned?

In terms of what can be drawn from these three case studies, environmental sampling and monitoring is not only about the numbers of organisms recovered; the type of species isolated can help with establishing origins and trending. The species need to be sense checked: isolates thus far only found in marine trenches or on the rim of volcanoes are unlikely to be recovered from cleanrooms. There is also a need, when batch disposition is resting on the identification, to draw on genotyping beyond the more conventional biochemical or proteomic techniques. An additional area where environmental monitoring can be useful is with moving sampling beyond the routine (to demonstrate a cleanroom remains in compliance); it can also serve as a powerful investigative tool to aid a deeper dive into investigations. This may include additional samples or sampling from areas that are not ordinarily assessed.

The case studies outlined are based on real-life events, collected across the pharmaceutical industry by the Pharmaceutical Microbiology Interest Group. 7 In reading such events there may be a sense of ‘this would not happen to me’; however, it is a recurrent criticism from regulatory inspectors that many investigations are not sufficiently thorough or fail to get to the actual root cause. These case studies may provide advice that can be applied to an organisation’s microbial investigation procedure.

About the author

Dr Tim Sandle  has over 25 years’ experience of microbiological research and biopharmaceutical processing. Tim is a member of several editorial boards and has authored 30 books on microbiology, healthcare and pharmaceutical sciences. Tim works for Bio Products Laboratory Limited (BPL) in the UK and is a visiting tutor at both the University of Manchester and UCL.

• McCullough K, Moldenhauer J. (2015) Introduction in Microbial Risk and Investigations. In McCullough, K. and Moldenhauer, J., (Eds) Microbial Risk and Investigations , Parenteral Drug Association (PDA) and Davis Healthcare International (DHI). Bethesda, MD, pp1-10
• Sutton S. (2011) Successful Microbial Investigations, American Pharmaceutical Review , 74 (2): 34-42
• Sandle T. (2014) Data Review and Analysis for Pharmaceutical Microbiology , Microbiology Solutions, UK., pp1-15
• Deng W, Burland V, Plunkett G, et al . (2002) Genome sequence of Yersinia pestis. Journal of Bacteriology . 184 (16): 4601–11
• Chanteau S, Rahalison L, Ralafiarisoa L, et al. (2003) Development and testing of a rapid diagnostic test for bubonic and pneumonic plague. Lancet 361: 211-216
• Sandle T. (2012): Sterility Test Failure Investigations, Journal of GxP Compliance , 16 (1): 1- 10
• Keen D. (2019) Contamination Case Studies, presented at Pharmig Irish Conference, 25th May 2019

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What if no root cause/probable cause found in microbial investigation in non-sterile product?

This is very informative, lots of useful information presented here.

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This case study focuses on their research which investigates the antibacterial activity of snake venom and it's potential as a source of novel antimicrobials.

Finding a new niche: mining for antibiotics in the nest of leafcutter ants

This case study is written by Professor Matt Hutchings, who is a Professor of Molecular Microbiology at the University of East Anglia and a member of the Microbiology Society. It focuses on finding anew species of Streptomyces to make new antibiotics.

SLIC: a novel technology to rapidly detect bacteria

This case study is written by Dr Robert Hammond, who is an Industrial Research Fellow at the University of St Andrews, and a member of the Microbiology Society. It focuses on using Scattered Light Integrating Collector (SLIC) technology to to rapidly detect bacteria.

Making antibiotics work better

This case study is written by Dr Andrew Edwards, Senior Lecturer in Molecular Microbiology at Imperial College London, UK. It focuses on ways to improve how antibiotics work.

Fighting Antimicrobial Resistance with Infection Prevention and Control: hospital drains and Carbapenemase-producing Enterobacterales

This case study is written by Dr Paz Aranega Bou, who is a water systems microbiologist at Public Health England (PHE) and a member of the Microbiology Society. It focuses on fighting Antimicrobial Resistance with Infection Prevention and Control: hospital drains and Carbapenemase-producing Enterobacterales

The antimicrobial properties of churchyard lichens

This case study is written by Dr Judith Taylor, who is a Lecturer at The Open University Graduate School (School of Environment, Earth & Ecosystem Sciences) and a member of the Microbiology Society. It focuses on the antimicrobial properties of churchyard lichens.

A 1000-year-old medieval remedy with anti-biofilm activity requires a combination of multiple ingredients

This case study is written by Dr Blessing Anonye (University of Central Lancashire) and Jessica Furner-Pardoe (University of Warwick). They are both members of the Microbiology Society and share their insights on the 1000-year-old medieval remedy with anti-biofilm activity, which requires a combination of multiple ingredients.

Development of a novel antibacterial nanocoating to reduce nosocomial infections

This case study is written by James Butler, who is a PhD student at the University of Plymouth and a member of the Microbiology Society. He discusses how he is working to help develop a novel antibacterial nanocoating to reduce nosocomial infections.

Mining deep-sea sponges for novel antibiotics

This case study is written by Matthew Koch, who is a PhD student at the University of Plymouth and a member of the Microbiology Society. It focuses on mining deep-sea sponges for novel antibiotics.

Investigating the possible role of L-form switching in recurrent urinary tract infections

This case study is written by Dr Katarzyna Mickiewicz, who is a Faculty of Medical Sciences Research Fellow at the University of Newcastle, UK, and a member of the Microbiology Society. It focuses on investigating the possible role of L-form switching in recurrent urinary tract infections.

Citizen science approaches to environmental sampling for AMR: #ScienceSolstice and #SummerSolstice

This case study is written by Jennifer Shelton, who is a final year PhD student at Imperial College London. It focuses on the Citizen Science approach to environmental sampling for antimicrobial resistance.

Building trust to improve antimicrobial stewardship

This case study is written by Dr Meher Rizvi, who is an Associate Professor at Sultan Qaboos University, Sultanate of Oman, and a member of the Microbiology Society. It focuses on building trust to improve antimicrobial stewardship.

Investigating the role of the human phageome in relation to AMR

This case study is written by Julie Callanan, who is a final year PhD student at the APC Microbiome Ireland SFI Research Centre, University College Cork, Ireland, and a member of the Microbiology Society. It focuses on investigating the role of the human phageome in relation to antimicrobial resistance (AMR).

Antimicrobial resistance in the environment

This case study is written by Dr Aimee Kaye Murray, who is a NERC Industrial Innovation Fellow and proleptic lecturer at the University of Exeter Medical School and a member of the Microbiology Society. It focuses on antimicrobial resistance in the environment.