Question

With regards to Pompe disease discuss the steps an accredited
laboratory will go through to reach a definitive diagnosis. In your answer
you should consider how the laboratory maintains the quality of their
service.

Answer 1

The primary role of a microbiology laboratory in patient care is to aid clinicians in identifying causative agents of various infectious diseases and help determine the antimicrobial susceptibility profiles, when appropriate.

These are the steps that to be consider for laboratory maintain and the quality:

Specimen Requirements:

Regardless of the specimen type, it is important for the laboratory to receive an adequate amount of specimen for testing. Unfortunately, it is not uncommon for the laboratory to receive a single swab with requests for viral, aerobic and anaerobic bacterial, fungal, and mycobacterial cultures. The yield of a culture is directly proportional to the amount of specimen initially inoculated onto the primary recover medium. Therefore, to obtain optimal results, a sufficient amount of specimen should be submitted. For example, when submitting an aspirate of an abscess, the clinician should submit more than a few drops of fluid or a swab for cultureWhen the clinician submits specimens to the laboratory for testing, all relevant information, such as the date and time of collection, type of specimen, and brief clinical scenario should be provided on the requisition form. The information is helpful for the laboratory to determine which growth medium to use to optimize growth. In certain cases in which a particularly virulent organism is suspected, the form should clearly indicate this to alert the laboratory technologist to handle the specimens with special safety measures and precautions, such as using a biologic safety cabinet. A poor-quality specimen is of no use to anyone and may yield unreliable results that could potentially affect patient care. Poor-quality specimens may produce false-negative results, because true pathogens may be overgrown by normal flora, and false-positive results; also, a commensal potential pathogen may be mistaken for the true causative agent of disease.

Blood:
Blood culture is the cornerstone of diagnosing many infectious diseases, and is of particular importance for determining the causative agent of endocarditis and other endovascular infections.2 Different methods and commercial systems are now available for detecting microorganisms. Generally, the blood culture system uses a broth-based medium that supports the growth of aerobic and anaerobic bacteria, depending on the bottle type used, and also some fungi, predominantly yeasts.
A major reason for failing to detect microorganisms in the bloodstream is an inadequate sampling of blood volume. When the recommended blood volume is inoculated (10 mL/bottle for an adult) into the blood culture bottles, most of the bacteremic or fungemic episodes can be recovered from two or three sets of blood cultures.2 The number of bottles used per draw and the number of draws per blood culture order can be confusing. A single blood culture order consists of two blood culture draws from different anatomic sites, preferably not through an IV catheter. Each of these draws, in turn, consists of filling two blood culture bottles with 10 mL of blood each; usually one aerobic and one anaerobic bottle comprise a blood culture set. It should be emphasized that the recommended volume of 20 to 30 mL is for each set of blood cultures, and that at least two sets are used for each blood culture order (i.e., four bottles total).There is little controversy when highly pathogenic microorganisms such as Staphylococcus aureus or Pseudomonas aeruginosa are isolated from the bloodstream. However, a single set of blood cultures with coagulase-negative staphylococci, Corynebacterium, or other gram-positive bacteria consistent with skin flora could represent blood culture contaminants or a true infection (i.e., the positive predictive value of a single blood culture draw is low). In other words, it is difficult to determine the clinical significance of such blood culture findings, because these organisms are part of the normal human skin flora.The concept is to provide more time for the more fastidious organisms to grow, thereby increasing the chances of recovery. This was classically used to detect the HACEK organisms (Haemophilus aphrophilus, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae), which were well-described causes of “culture-negative” endocarditis. However, the newer blood culture system technology and improvements in blood culture media have resulted in recovery rates for these organisms that are almost as good as those for more common pyogenic bacteria, such as Staphylococcus aureus.3 Nevertheless, some will still extend incubation or perform terminal subcultures if a fastidious pathogen is a suspected cause of endocarditis. In our experience, we have seen culture and biopsy-proven cases of Propionibacterium acnes endocarditis that grew only after extended incubation.

Sputum and Other Respiratory Tract Specimens:
Diagnosing lower respiratory tract infections and pneumonia remains complicated and requires a combination of clinical and laboratory findings for optimal diagnosis. Expectorated sputum, even with its own limitations, remains the most common and easiest specimen to obtain for diagnosis. However, when a sputum sample is submitted to the microbiology laboratory for routine bacterial culture and stain, clinicians should be familiar with some of the issues regarding this type of specimen.4 The specimen is first smeared onto a glass slide and Gram staining is performed as a screen. A high-quality purulent sputum specimen should contain many neutrophils and only relatively few epithelial cells. If many epithelial cells are seen—specifically if more than 25 epithelial cells are seen per low-power field—the specimen is contaminated with upper oropharyngeal flora (i.e., saliva) and may not even be from the lower respiratory tract. In such a case, even if microorganisms are seen on Gram staining, it cannot be ascertained whether the organism in question is a pathogen or simply part of the normal upper respiratory tract flora.Finally, it is prudent to remember that Gram staining is only a screening tool to aid clinicians in diagnosis. The classic causes of atypical pneumonia, Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella species, do not stain (M. pneumoniae and C. pneumoniae) or do not stain well (Legionella) with Gram staining. The diagnosis of infections by these agents uses other methodologies such as serology testing, antigen detection, polymerase chain reaction (PCR) assay, or a combination of these. An astute clinician should have a high index of suspicion for pneumonia if the history, physical examination, and radiograph are suggestive of such an infection, even if the Gram staining result is nondiagnostic or negative for microorganisms, and appropriate empirical antimicrobial treatment should be given.

Stool:
Patients with diarrheal illness could have a spectrum of clinical presentations, ranging from mild, self-limited, loose stools, with minimal to moderate abdominal discomfort, to severe abdominal pain, with or without bloody stool. Many bacterial, viral, and parasitic organisms are capable of causing gastrointestinal (GI) infections. It is beyond the scope of this chapter to cover this exhaustive list and many excellent texts and references are available to discuss these pathogens more fully. However, some key concepts should be emphasized to help the clinician obtain maximal laboratory data to make an accurate diagnosis and treat patients appropriately. The laboratory, at the same time, is challenged to perform cost-effective and efficient assays while helping clinicians achieve their diagnostic goals..
Diarrheal illness generally can be divided anatomically into those that predominantly affect the small intestine and those that affect the large intestine. Pathogens that affect the small intestine usually cause a malabsorptive type of symptoms, with loose watery stools. Some classic examples include Giardia lamblia and Cryptosporidium parvum infections. Infections that affect the large intestine usually manifest with diarrhea caused by colitis that may be bloody; these patients may be toxic or even develop septic shock if the infection is severe enough. Some large intestinal pathogens include Salmonella, Shigella, Campylobacter, and Clostridium difficile.The definitive diagnosis of C. difficile infection is made by the detection of the toxins—namely, toxins A and B in the stools of patients suspected of having this disease. The toxins can be detected by enzyme-linked immunoassay (EIA) but, traditionally, the tissue culture method is used for the detection of toxin B. With this method, a cytopathic effect (CPE) is produced that may be blocked using antitoxin. In many laboratories, combined toxins A and B EIAs have replaced tissue culture, generally because they have a more rapid turnaround time and are less labor- and resource-intensive. However, it should be noted that EIA has a sensitivity of only 75% to 85% and a strong clinical suspicion should be entertained, even if there is a negative C. difficile toxin EIA result.

Wound, Wound Drainage, and Cerebrospinal Fluid:
Management of wound culture presents some difficult challenges to the microbiology laboratory. Many issues need to be addressed in regard to these types of cultures:
   What is the appropriate method to collect specimens?
   Do swabs have any roles in bacterial cultures?
   What is the clinical significance of cultures that contain mixed flora?
Should wound drainage (e.g., Jackson-Pratt drain) be cultured routinely to rule out active infections?
These are some questions that should be carefully considered before submitting specimens to the laboratory.
The most effective way to treat an abscess or infected fluid collection is to drain the abscess in conjunction with antimicrobial therapy. Rarely, an abscess can be cured just by medical treatment. Occasionally, fluid to be collected is located in a deep structure in which percutaneous or even surgical drainage is deemed too risky for the patient. In such cases, prolonged antimicrobial therapy is the only viable option and these cases should be followed closely to assess clinical response. In some other cases, a radical surgical procedure might be the best option for managing chronic infection. For example, patients with chronic empyema who have undergone multiple surgical procedures for decortication may be best managed using a Clagett open-window thoracostomy for drainage.
The appropriate assessment of cerebrospinal fluid (CSF) is absolutely crucial in managing patients with suspected cases of meningitis or encephalitis. The basic tenet for collecting CSF is similar to that for other specimens, but particular attention should be given to the aseptic technique used during collection. Before performing a lumbar puncture, the skin should be thoroughly cleansed with iodophor or chlorhexidine. The patient should preferably be lying in a recumbent position; occasionally, for obese patients it might be easier to obtain the fluid by asking the patient to sit upright and hunch forward to better accentuate the L4-L5 interspace. However, in such a position, the opening pressure cannot be accurately measured. In certain central nervous system infections, an assessment of the initial opening pressure is absolutely crucial for managing the patient. For example, a patient with Cryptococcus neoformans meningitis may have an elevated intracranial pressure (ICP), with few or subtle clinical signs and symptoms. Knowing the patient’s baseline ICP will assist clinicians and neurosurgeons regarding the performance of shunting, if necessary.
   The quantity of CSF is especially crucial and as much fluid as possible should be obtained, because multiple tests are usually ordered and some tests, such as CSF for cytology, require a large amount of sample. For certain pathogens, such as fungi and mycobacterial species, the microbial recovery is directly proportional to the volume of sample used to inoculate the cultures. Therefore, at least 10 to 20 mL of CSF should be submitted to the laboratory for testing. The clinical findings should dictate the type of testing to be performed.

Viral Cultures:
Viral diseases are caused by a heterogeneous mixture of viruses and, depending on each virus’s unique pathogenesis, the clinician needs to submit the appropriate specimen for testing. Many methodologies can be used for diagnosing viral diseases, such as serologic (antibody and antigen testing), viral culture, and molecular methods, such as the PCR assay and related techniques. Diagnostic virology will not be discussed in detail here; this section will focus specifically on viral cultures.
Molecular methods such as PCR have gained popularity and acceptance in recent years. This methodology is indispensable, especially in the field of clinical virology, One major drawback is the cost of running a molecular assay. Typically, tests using molecular methods are more expensive than conventional viral cultures. Consequently, viral cultures are still being used in many laboratories, especially small hospitals and laboratories in which the volume of testing might be low, and cost containment is important.alcium alginate can affect the recovery of herpes simplex and varicella zoster virus.10 If the specimens cannot be processed in a timely fashion, the general rule for keeping the virus viable is to refrigerate the specimens. The clinician should check with the laboratory regarding the specific types of virus collection, transport media, and storage options before obtaining specimens.