Laboratory Diagnosis of Infection Due to Bacteria, Fungi, Parasites, and Rickettsiae

Specimen Collection

Many advances have been made to maximize the recovery of bacteria from blood and to minimize contamination during specimen collection. Blood cultures collected from venipuncture sites following antisepsis with chlorhexidine gluconate and 70% isopropyl alcohol have lower contamination rates than from sites following antisepsis with povidone-iodine. Following antisepsis, sites must be allowed to dry for at least 1 minute before venipuncture. Because of limited safety data, chlorhexidine is not recommended for use in infants <2 months; instead, povidone-iodine is preferred in this age group. However, in one study, the use of 1% chlorhexidine was not only safe in newborns ≥1500 grams body weight but was more effective in reducing contamination rates. The rubber septa of blood culture bottles or tubes should be disinfected using 70% isopropyl or ethyl alcohol prior to insertion of the needle into the bottle.

The quantity of blood cultured and the proportion of blood to broth are extremely important factors in blood cultures; a ratio of >1:5 is desirable. Inadequate specimen volume is the leading cause of low blood culture sensitivity. Most blood culture broths contain an anticoagulant, sodium polyanetholesulfonate (SPS), ranging in concentration from 0.025%–0.05%, which inhibits phagocytosis and the bactericidal activity of serum, inactivates complement, and neutralizes lysozyme and aminoglycosides. SPS can inhibit the growth of some micro-organisms, including Neisseria spp., Streptobacillus moniliformis, and Francisella tularensis. An appropriate ratio of blood to broth volume dilutes SPS, thereby decreasing the natural inhibitory factors present in the blood, and also dilutes antimicrobial agents if they were present in the patient’s bloodstream. Many older blood culture systems were suboptimal for pediatric use because the 5- to 10-mL recommended blood volume was inappropriate for infants. Bacteremia in children usually is quantitatively higher than in adults; 1–5 mL may be an adequate sample from children, whereas higher volumes may be needed from adults. Adequate blood volume per bottle not only increases recovery yields but also decreases time to detection. BACTEC Peds Plus/F medium is specifically formulated to optimize recovery of microorganisms from 1–3 mL of blood. A supplement (BACTEC FOS Culture Supplement) is available for enhancement of the recovery of Haemophilus spp. and Neisseria spp. Table 286.5 shows recommended blood volumes according to body weight. Although a single sampling may be sufficient for many patients with bacteremia, multiple samples are appropriate in certain circumstances (e.g., in suspected endocarditis, when two to three samples are desirable). The total volume of blood is probably more important in this circumstance than the number of samples. At least two sets of cultures are helpful in children with indwelling intravascular catheters. Bottles should not be inoculated with more than the manufacturer’s recommended amount.

Once blood is inoculated into a broth bottle, rapid incubation is essential. Delays in insertion of bottles into automated, continuously-monitored blood culture systems can result in a delay in detecting the presence of bacteremia.

Media

Advances in blood culture systems have increased the yield of blood cultures, reduced the time to organism recovery, and diminished laboratory technologist hands-on time. In addition, some systems were developed to maximize recovery of fastidious organisms. Examples of these systems include the Isolator (Wampole), a manual blood culturing system that utilizes blood cell lysis and centrifugal concentration prior to direct inoculation of the microbial pellet onto agar, and the continuous-monitoring blood culture systems, which include the VersaTREK (Thermo Scientific), BacT/ALERT (bioMérieux), and BACTEC (Becton Dickinson and Company) systems. For the latter three systems, several broth formulations are available, including media for the recovery of aerobic and anaerobic bacteria, mycobacteria, and fungi. BACTEC Peds Plus/F medium is specifically formulated to recovery organisms from small blood volumes and optimize recovery of fastidious organisms. It should be noted that no single system is optimal for recovery of all micro-organisms.

The Isolator system consists of a sterile tube (1.5 or 10 mL) that contains saponin and SPS, which lyse red and white blood cells and inactivate complement and immunoglobulins, respectively. Hemoglobin binds to SPS and prevents inhibition of bacteria. The larger tubes are centrifuged at 300 g for 30 minutes in a fixed-angle rotor. With the use of an Isostat press and specialized pipettes, the lysate is removed and the microbial pellet is retrieved and inoculated directly onto agar media, which are incubated and examined daily for growth. Media can be selected to maximize the recovery of suspected pathogens. ^, In addition, isolation of certain fungi is enhanced. As little as 1 mL of blood is required, and direct agar inoculation allows for quantification of bacteria. Excess contamination can occur with this system; however, careful handling and inoculation of agar within a biological safety cabinet decreases the likelihood.

Compared with conventional systems, BACTEC has demonstrated increased recovery of organisms and decreased time to detection, particularly for Mycobacterium spp . The BD BACTEC FX blood culture system is a continuous monitoring, “noninvasive” blood culture system that uses internal fluorescent CO ₂ sensors to detect the metabolic activity of bacteria growing within the culture broth. BACTEC Plus Aerobic/F, Plus Anaerobic/F, and Peds Plus/F bottles contain resins that may decrease the inhibitory effect of antibiotics present within the blood. This system can assist clinicians in identifying an intravascular catheter-related infection by utilizing a differential time-to-positivity between a positive culture result from an intravascular catheter site and a peripheral venipuncture site when equal volumes were collected, concurrently.

BacT/ALERT and VersaTREK systems are two additional continuous monitoring blood culture systems that have been demonstrated to be useful for the detection of bacteremia and fungemia, with a higher yield of recovery and a reduction in time-to-detection compared with manual blood culture systems.

Incubation

Older “traditional” broth bottles were incubated for 7 days. Bottles were inspected for macroscopic growth daily and subcultured blindly. Acridine orange staining was performed after 18–24 hours of incubation. With newer, automated systems, bottles are incubated at 35°C with constant agitation and continuous monitoring, usually for 5 days prior to reporting and discarding negative cultures.

Most pediatric pathogens are isolated within 72 hours of incubation. With the newer blood culture systems, most bacterial pathogens are detected within 48 hours. Incubation for >5 days is not usually warranted except in certain situations (e.g., infection suspected due to fungi, Bartonella henselae, and a few others).

Special Pathogens and Situations

Clinicians must communicate the suspicion of brucellosis, tularemia, melioidosis, glanders, anthrax, plague, meningococcal sepsis/meningitis, and enteric fever to the laboratory so that enhanced precautions can be taken when manipulating these cultures. If brucellosis is suspected, the automated blood culture is held for a minimum of 7 but no more than 10 days. Subculture to a blood agar, chocolate agar, or brucella agar plate and incubation in 5%–10% CO ₂ at 37°C for at least 72 hours is usually adequate. For the isolation of cell wall-deficient bacteria, hypertonic medium (containing 10% sucrose or mannitol) is required. Nutritionally deficient organisms, such as Abiotrophia spp., should be suspected when organisms are evident on Gram stain but fail to grow when subcultured. Broth can be subcultured onto a blood agar plate streaked with Staphylococcus aureus, or media can be supplemented with 0.001% pyridoxal hydrochloride and 0.05%–0.1% L-cysteine. A pyridoxal-impregnated disk also can be used. Plates are usually incubated overnight at 35°C–37°C in 5%–10% CO ₂ . Although rare causes of bacteremia and endocarditis, the HACEK group of micro-organisms, Haemophilus parainfluenzae , Aggregatibacter aphrophilus, A. paraphrophilus, A. actinomycetemcomitans, Cardiobacterium spp. , Eikenella corrodens, and Kingella kingae , grow within the routine incubation period for automated blood culture systems.

Special conditions are required for isolation of Bartonella spp. and should only be performed by laboratories familiar with culture and identification of this organism. The Isolator system is often used, with subsequent inoculation onto media enriched with fresh blood (5% rabbit blood) and incubated in CO ₂ at 35°C–37°C for 15 days.

Studies have demonstrated the ability of 16S rRNA gene polymerase chain reaction (PCR) to detect bacteremia within hours of collecting a blood specimen. Multiplex blood PCR can detect the presence of gram-positive and gram-negative bacteria and fungi in febrile neutropenic patients. PCR was capable of diagnosing and serotyping pneumococcal bacteremic community-acquired pneumonia in children. In persons with S. aureus bacteremia, PCR identified those patients with methicillin-susceptible S. aureus, permitting reduction in vancomycin use, hospital stays, and costs. For rapid identification of most common bloodstream pathogens and antimicrobial resistance genes directly from positive blood culture broths, a number of US Food and Drug Administration (FDA)-cleared methods are commercially available and include peptide-nucleic acid fluorescence in situ hybridization (PNA-FISH), multiplex nucleic acid amplification, nanoparticle probe technology, and mass spectrometry.

Specimen Collection and Processing

CSF must be transported to the laboratory without delay because CSF is hypotonic and bacterial and human cells can lyse (thereby affecting the cell count and contributing to falsely abnormal biochemical analysis) or utilize glucose and thus lower measured levels. At room temperature, cell counts decrease approximately 32% in 1 hour and 50% in 2 hours after collection. Neutrophils are affected more than lymphocytes. Refrigeration can render fastidious bacteria nonviable. If delay is expected, samples are stored at room temperature or incubated at 35°C–37°C to maximize isolation.

Routinely, CSF should be inoculated onto sheep blood agar, chocolate agar, and enrichment broth such as thioglycolate broth and incubated for up to 4 days at 37°C in 5%–10% CO ₂ . If Gram stain is positive but culture demonstrates no growth at 72 hours, the culture is held for an additional 4 days. The minimal volume acceptable for culture of fungi and Mycobacterium is 2 mL; 10–15 mL is preferred.

Centrifugation by cytospinning (2000 rpm; 350 g ) to maximize pellet formation of bacteria and cellular elements produces a yield on direct-smear examination of CSF by Gram stain superior to that of unconcentrated samples. Leukocyte morphology is preserved, and examination of large numbers of cells improves the validity of the differential cell count. In one study, smears were positive for bacteria in 78% of samples when cytospinning was performed compared with 56% for unconcentrated samples.

Gram stain demonstrates organisms in 75%–90% of untreated patients with meningitis. Patients with meningitis due to Streptococcus pneumoniae and Haemophilus influenzae infection are more likely to have a positive Gram stain (90% and 86%, respectively) than due to Neisseria meningitidis (75%). The yield of Gram stain correlates with the density of bacteria in CSF (e.g., positivity in 97% of infections with ≥10 ⁵ CFU/mL of bacteria and only 25% with <10 ³ CFU/mL). The yield of Gram stain decreases in patients receiving antimicrobial therapy, even orally.

Acridine orange, or 3,6- bis (dimethylamino)acridine hydrochloride, staining is a more sensitive technique than Gram stain for detecting bacteria, especially in patients who have received antimicrobial therapy. ^,

Multiplex PCR that enables the simultaneous detection of numerous common bacterial, viral, and fungal agents associated with meningitis and encephalitis has become commonplace in clinical laboratories throughout the world. The FilmArray Meningitis/Encephalitis (ME) Panel (Biofire Diagnostics, Salt Lake City, UT) is an FDA-cleared, rapid multiplex PCR system that has proven to be a valuable tool for the diagnosis of bacterial meningitis, especially in patients who have received antibiotics. ^, Multiple studies have demonstrated its clinical usefulness, with sensitivities similar to routine culture technology but with a shorter turnaround time. Isolation of an organism from culture permits antimicrobial susceptibility testing. PCR appears in some studies to have a higher sensitivity than bacterial culture. In one study, 16S rRNA gene PCR had an overall sensitivity of 93%, with a specificity of 98%. In addition, the technology is being used in outbreak investigations. In a recent study, investigators were able to link 3 detected cases of Streptococcus salivarius meningitis to a specific healthcare provider.

Bacterial antigen detection is usually not useful in the diagnosis and management of meningitis. Value, if any, appears to be limited to patients whose Gram stain, CSF culture, and blood culture are negative; pretreated patients; and when lumbar puncture is postponed because of the severity of illness. ^, While PCR appears to be a superior detection method, in some countries with limited resources, the combination of culture, Gram stain, and antigen detection has demonstrated a very high sensitivity in identifying causative pathogens.

Specimen Collection

Tables 286.1 and 286.2 highlight proper collection of respiratory tract specimens for bacterial isolation. Throat swab for the detection of Streptococcus pyogenes (i.e., group A Streptococcus [GAS]) is the most common respiratory tract specimen sent for culture; proper collection of the sample (by swabbing of the tonsillar surface, posterior pharyngeal wall, and opposite tonsillar surface while avoiding the tongue and saliva) affects the yield of the culture.

Rapid Tests for Group A Streptococcus

Routine culture on agar requires 24–48 hours for results and could delay therapy for some patients. Rapid detection assays for streptococcal antigen can yield results in 10–70 minutes. The specificity of various tests is 62%–100%, but the sensitivity is lower than culture. Although initial studies performed in microbiology laboratories demonstrated high sensitivity, performance in other settings shows variable results. Results are influenced by the skill, experience, and expertise of the person obtaining the throat swab and performing the assay. In a community office-based study, the sensitivity of the office culture for GAS was found to be higher than the rapid antigen-detection test (81% versus 70%). Both tests have specificities greater than 97%. Tests utilizing optical immunoassay have consistently demonstrated sensitivities and specificities higher than other assays. The isolation of GAS on agar permits antimicrobial susceptibility testing for macrolide resistance.

A reflex culture has been recommended for individuals with a negative rapid antigen test. Since specificities are high, positive antigen tests do not need to be confirmed by culture. ^, Although sensitivity increases with severity of illness, the sensitivity of the assay may not be high enough to avoid performance of a culture.

Most methods that use DNA probes and real-time PCR have demonstrated high specificity and good sensitivity. ^, Unfortunately, many of these tests are difficult to perform at the site of patient care. The cobas Liat PCR system (Roche Diagnostics) and others have been cleared by the FDA for diagnosis of GAS infection. Among the many benefits of these systems include their short time to detection and ability to act as a stand-alone test for diagnosis of GAS infection; confirmatory testing (i.e., culture or PCR) is not necessary. Loop-mediated isothermal amplification (LAMP) assays for detection of GAS are also commonplace and offer high sensitivity and high specificity and also eliminate the need for antigen and reflex culture testing. Some newer technology permits the detection of macrolide and clindamycin resistance.

Antigen detection assays have been used to detect the presence of GAS at extrapharyngeal sites such as in pyogenic arthritis, cellulitis, and parapneumonic effusions. One kit demonstrated high sensitivity and specificity for detecting antigen at skin sites. Antigen detection and PCR assays have enhanced the detection of respiratory pathogens in parapneumonic effusion/empyema.

Special Specimens

Tympanocentesis plus sinus aspiration for culture is extremely useful in special situations (e.g., immunocompromised patients, patients with intracranial complications, and those who fail to respond to antimicrobial therapy). Data are conflicting regarding the validity of culture of the nasopharynx in predicting the pathogens of sinusitis and otitis media. Routine use is not indicated.

Collection of sputum from children with lower respiratory tract infections is technically difficult. Aspiration of deep pharyngeal/tracheal secretions (with a Leuken trap) is used by some, and tracheal aspiration is used commonly in intubated patients and in those who have undergone tracheostomy with tube placement. In older children, sputum is a valuable specimen. The presence of ≥10 squamous epithelial cells per low-power field is highly suggestive of an oropharyngeal site of specimen origin. Conversely, the presence of >25 white blood cells per low-power field denotes an adequate specimen. Organism isolation at quantities ≥10 ⁴ CFU/mL is predictive of the pathogen of infection, as proved by biopsy. Gram stain should be used to aid in the interpretation of isolates from culture.

Transtracheal aspiration is technically difficult in children and is seldom performed. Bronchoscopy, and especially a quantitative bronchoalveolar lavage specimen or protected brush, is useful in the diagnosis of Pneumocystis jirovecii , mycobacterial, fungal, and bacterial infections, as well as for molecular and antigen testing for a variety of pathogens.

Burkholderia cepacia Complex and Burkholderia gladioli

Isolation of B. cepacia complex organisms (e.g., Burkholderia multivorans ) and B. gladioli from the sputum of patients with cystic fibrosis requires the use of selective media. B. cepacia -selective agar (BCSA) or oxidative-fermentative base-polymyxin B-bacitracin-lactose (OFPBL) medium have been used. ^, It appears that BCSA may be superior to OFPBL.

Bordetella species

Currently available diagnostic tests for Bordetella have variable sensitivity, depending on the case definition, level of immunization, adequacy of collection and transport, and diagnostic method. ^, When properly performed, culture is superior to direct immunofluorescence assay on nasopharyngeal secretions. Culture is more likely to be positive during the first 2 weeks of illness than later. Serologic tests by enzyme immunoassay (EIA; B. pertussis -specific immunoglobulin G) are sensitive but may be difficult to interpret and usually require acute and convalescent sera. PCR assays may provide higher sensitivity with a quicker turnaround. In addition, in persons with symptoms for ≥2 weeks, PCR demonstrates a higher sensitivity than culture or DFA. ^{, ,} A combination of culture, serology, and PCR provides a greater sensitivity when performed on previously immunized individuals with cough illness. ^, In many diagnostic clinical microbiology laboratories, PCR targeting the repetitive elements IS 481 and US 1001 of Bordetella pertussis and B. parapertussis , respectively, is the test of choice, and fluorescent staining and culture are no longer performed. Standardization of PCR assay and false-positive results are problematic. The Centers for Disease Control and Prevention (CDC) recommendations for primers and test conditions are available at https://www.cdc.gov/vaccines/pubs/surv-manual/chpt22-lab-support.html .