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- Parasite Classification | - Parasite, Body Site |
- Stool Testing Order Recommendations | - STAT Testing |
- Fecal Fixatives | - Stool Collection Options | - Report Comments |
- Tips for Fecal ImmunoAssay | - Malaria (5 Species) |
- Malaria (5 Species) Images | - Rapid Malaria Testing |
- Malaria Parasitemia Method | - Malaria Parasitemia Interpretation |
- USE OF A REFERENCE LABORATORY FOR PARASITE BLOOD DIAGNOSTIC TESTING (Including the Binax Rapid Test and Report Comments)

- HELMINTH PARASITES ASSOCIATED WITH EOSINOPHILIA | - Histology: Staining Characteristics - Table 1 | - Histological Identification of Parasites - Table 2 | - Microscope Calibration | - Figures for Histology Identification Table 2
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STAT TESTING IN PARASITOLOGY

STAT REFERS TO ORDERING, COLLECTION, PROCESSING, TESTING, AND REPORTING

RISK MANAGEMENT ISSUES ASSOCIATED WITH STAT TESTING

There are two circumstances in diagnostic medical parasitology that represent true STATs. One is a suspected case of primary amebic meningoencephalitis (PAM) caused by Naegleria fowleri or granulomatous amebic encephalitis (GAE) caused by Acanthamoeba spp., Balamuthia mandrillaris, or Sappinia diploidea, and the other situation is any case where thick and thin blood films are requested for testing for blood parasites, possibly those that cause malaria and/or babesiosis. Extensive discussions of these organisms can be found in the following reference (L. S. Garcia, Diagnostic Medical Parasitology, 6th ed., ASM Press, Washington, DC, 2016).

REQUEST FOR EXAMINATION OF SPECIMENS FOR FREE-LIVING AMEBAE

Primary amebic meningoencephalitis (PAM) caused by N. fowleri is an acute, suppurative infection of the brain and meninges. With extremely rare exceptions, the disease is rapidly fatal in humans. The period between contact with the organism and onset of clinical symptoms such as fever, headache, and rhinitis may vary from 2 to 3 days to as long as 7 to 15 days. The amebae may enter the nasal cavity by inhalation or aspiration of water, dust, or aerosols containing the trophozoites or cysts. The organisms then penetrate the nasal mucosa, probably through phagocytosis of the olfactory epithelium cells, and migrate via the olfactory nerves to the brain. Data suggest that N. fowleri directly ingests brain tissue by producing food cups or amebostomes, in addition to producing a contact-dependent cytolysis which is mediated by a heat- stable hemolytic protein, heat-labile cytolysis, and/or phospholipase enzymes. Cysts of N. fowleri are generally not seen in brain tissue.

Early symptoms include vague upper respiratory distress, headache, lethargy, and occasionally olfactory problems. The acute phase includes sore throat, stuffy blocked or discharging nose, and severe headache. Progressive symptoms include pyrexia, vomiting, and stiffness of the neck. Mental confusion and coma usually occur approximately 3 to 5 days prior to death. The cause of death is usually cardiorespiratory arrest and pulmonary edema.

PAM can resemble acute purulent bacterial meningitis, and these conditions may be difficult to differentiate, particularly in the early stages. The CSF may have a predominantly polymorphonuclear leukocytosis, increased protein concentration, and decreased glucose concentration like that seen with bacterial meningitis. Unfortunately, if the CSF Gram stain is interpreted incorrectly (identification of bacteria as a false positive), the resulting antibacterial therapy has no impact on the amebae and the patient usually dies within several days.

Extensive tissue damage occurs along the path of amebic invasion; the nasopharyngeal mucosa shows ulceration, and the olfactory nerves are inflamed and necrotic. Hemorrhagic necrosis is concentrated in the region of the olfactory bulbs and the base of the brain. Organisms can be found in the meninges, perivascular spaces, and sanguinopurulent exudates.

Clinical and laboratory data usually cannot be used to differentiate pyogenic meningitis from PAM, so the diagnosis may have to be reached by a process of elimination. A high index of suspicion is often mandatory for early diagnosis. All aspects of diagnostic testing (specimen collection, processing, examination, and reporting) should be considered STAT. Although most cases are associated with exposure to contaminated water through swimming or bathing, this is not always the case. The rapidly fatal course of 3 to 6 days after the beginning of symptoms (with an incubation period of 1 day to 2 weeks) requires early diagnosis and immediate chemotherapy if the patient is to survive.

Analysis of the CSF shows decreased glucose and increased protein concentrations. Leukocyte counts may range from several hundred to >20,000 cells per mm3. Gram stains and bacterial cultures of CSF are negative; however, the Gram stain background can incorrectly be identified as bacteria, thus leading to incorrect therapy for the patient.

A definitive diagnosis could be made by demonstration of the amebae in the CSF or in biopsy specimens. Either CSF or sedimented CSF should be placed on a slide under a coverslip and observed for motile trophozoites; smears can also be stained with Wright’s or Giemsa stain. CSF, exudate, or tissue fragments can be examined by light microscopy or phase-contrast microscopy. Care must be taken not to mistake leukocytes for actual organisms or vice versa. It is very easy to confuse leukocytes and amebae, particularly when one is examining CSF by using a counting chamber, hence the recommendation to use just a regular slide and coverslip. Motility may vary, so the main differential characteristic is the spherical nucleus with a large karyosome.

Specimens should never be refrigerated prior to examination. When centrifuging the CSF, low speeds (250 x g) should be used so that the trophozoites are not damaged. Although bright-field microscopy with reduced light is acceptable, phase microscopy, if available, is recommended. Use of smears stained with Giemsa or Wright’s stain or a Giemsa-Wright’s stain combination can also be helpful. If N. fowleri is the causative agent, only trophozoites are normally seen. If the infecting organism is Acanthamoeba spp., cysts may also be seen in specimens from individuals with central nervous system (CNS) infection. Unfortunately, most cases are diagnosed at autopsy; confirmation of these tissue findings must include culture and/or special staining with monoclonal reagents in indirect fluorescent-antibody procedures. Organisms can also be cultured on nonnutrient agar plated with Escherichia coli (a food source for the amebae).

In cases of presumptive pyogenic meningitis in which no bacteria are identified in the CSF, the computed tomography appearance of basal arachnoiditis (obliteration of basal cisterns in the precontrast scan with marked enhancement after the administration of intravenous contrast medium) should alert the staff to the possibility of acute PAM.

The amebae can be identified in histologic preparations by indirect immunofluorescence and immunoperoxidase techniques. The organism in tissue sections looks very much like an Iodamoeba bütschliitrophozoite, with a very large karyosome and no peripheral nuclear chromatin; the organisms can also be seen with routine histologic stains.

Requests for examination of specimens for the other free-living amebae (Acanthamoeba, Sappinia, Balamuthia) should also be considered STAT requests.

GRANULOMATOUS AMEBIC ENCEPHALITIS AND AMEBIC KERATITIS

The most characteristic feature of Acanthamoeba spp. is the presence of spine-like pseudopods called acanthapodia. Several species of Acanthamoeba (A. culbertsoni, A. castellanii, A. polyphaga, A. astronyxis,
A. healyi, and A. divionensis) cause GAE, primarily in immunosuppressed, chronically ill, or otherwise debilitated persons. These patients tend to have no relevant history involving exposure to recreational freshwater. Acanthamoeba spp. also cause amebic keratitis, and it is estimated that to date approximately 1,000 cases of Acanthamoeba keratitis have been seen in the United States.

GAE caused by freshwater amebae is less well defined and may occur as a subacute or chronic disease with focal granulomatous lesions in the brain. The route of CNS invasion is thought to be hematogenous, with the primary site being the skin or lungs. In this infection, both trophozoites and cysts can be found in the CNS lesions. An acute-onset case of fever, headache, and pain in the neck preceded by 2 days of lethargy has also been documented. The causative organisms are probably Acanthamoeba spp. in most cases, but it is possible that others are involved such as Balamuthia mandrillaris and Sappinia diploidea.

Cases of GAE have been found in chronically ill or immunologically impaired hosts; however, some patients apparently have no definite predisposing factor or immunodeficiency. Conditions associated with GAE include malignancies, systemic lupus erythematosus, human immunodeficiency virus (HIV) infection, Hodgkin’s disease, skin ulcers, liver disease, pneumonitis, diabetes mellitus, renal failure, rhinitis, pharyngitis, and tuberculosis. Predisposing factors include alcoholism, drug abuse, steroid treatment, pregnancy, systemic lupus erythematosus, hematologic disorders, AIDS, cancer chemotherapy, radiation therapy, and organ transplantation. This infection has become more widely recognized in AIDS patients, particularly those with a low CD4+ cell count.
Laboratory examinations similar to that for N. fowleri can be used to recover and identify these organisms; the one exception is recovery by culture, which has not proven to be as effective with GAE patients infected with B. mandrillaris. These tests should also be considered STAT.

REQUEST FOR BLOOD FILM EXAMINATION

Malaria is one of the few parasitic infections considered to be immediately life-threatening, and a patient with the diagnosis of P. falciparum malaria should be considered a medical emergency because the disease can be rapidly fatal. Any laboratory providing the expertise to identify malarial parasites should do so on a STAT basis (24 h/day, 7 days/week).

Patients with malaria can present for diagnostic blood work when they are least expected. Laboratory personnel and clinicians should be aware of the STAT nature of such requests and the importance of obtaining some specific patient history information. On microscopic examination of the blood films, the typical textbook presentation of various Plasmodium morphologies may not be seen by the technologist. The smears should be examined at length and under oil immersion. The most important thing to remember is that even though a low parasitemia may be present on the blood smears, the patient may still be faced with a serious, life-threatening disease.

It is important for both physicians and laboratorians within areas where malaria is not endemic to be aware of the problems associated with malarial diagnosis and to remember that symptoms are often nonspecific and may mimic other medical conditions. Physicians must recognize that travelers are susceptible to malarial infection when they visit a country where malaria is endemic, and that they should receive prophylactic medication.

With the tremendous increase in the number of people traveling from the tropics to malaria-free areas, the number of imported malaria cases is also on the rise. There have been reports of imported infected mosquitoes transmitting the infection among people who live or work near international airports. It is also possible that mosquitoes can reach areas far removed from the airports. This situation has been
termed ‘‘airport malaria,’’ malaria that is acquired through the bite of an infected anopheline mosquito by persons with apparently no risk factors for the disease. Unfortunately, unless a careful history is obtained, the diagnosis of malaria can be missed or delayed. Tests to exclude malaria should be considered for patients who work or live near an international airport and who present with an acute febrile illness. The potential danger of disseminating the mosquito vectors of malaria via aircraft is well recognized; however, modern disinfection procedures have not yet eliminated the risk of vector transportation. Not only can insects survive nonpressurized air travel, but they may be transported further by car or other means after arriving at the airport.

We usually associate malaria with patients having a history of travel within an area where malaria is endemic. However, other situations that may result in infection involve the receipt of blood transfusions, use of hypodermic needles contaminated by prior use (for example, by drug addicts), possibly congenital infection, and transmission within the United States by indigenous mosquitoes that acquired the parasites from imported infections.

Frequently, for a number of different reasons, organism recovery and subsequent identification are more difficult than the textbooks imply. It is very important that this fact be recognized, particularly when one is dealing with a possibly fatal infection with P. falciparum. It is important to ensure that clinicians are familiar with the following issues.

Automated Instrumentation

Potential diagnostic problems with the use of automated differential instruments have been reported. Some cases of malaria, as well as Babesia infection, have been completely missed by these methods. The number of fields scanned by a technologist on instrument-read smears is quite small; thus, failure to detect a low parasitemia is almost guaranteed. In cases of malaria and Babesia infection, after diagnosis had been made on the basis of smears submitted to the parasitology division of the laboratory, all previous smears examined by the automated system were reviewed and found to be positive for parasites. Failure to make the diagnosis resulted in delayed therapy. These instruments are not designed to detect intracellular blood parasites, and the inability of the automated systems to discriminate between uninfected erythrocytes and those infected with parasites may pose serious diagnostic problems in situations where the parasitemia is ≤0.5%.

 

Patient Information

When requests for malarial smears are received in the laboratory, some patient history information should be made available to the laboratorian. This history can be obtained by asking the ordering physician important questions such as the following:

  1. Where has the patient been, and what was the date of return to the United States? (Where do you live and where do you work? [‘‘airport malaria’’])
  2. Has malaria ever been diagnosed in the patient before? If so, which species was identified?
  3. What medication (prophylaxis or otherwise) has the patient received, and how often? When was the last dose taken?
  4. Has the patient ever received a blood transfusion? Is there a possibility of another needle transmission (drug user)?
  5. When was the blood specimen drawn, and was the patient symptomatic at the time?
  6. Is there any evidence of a fever periodicity?

Answers to such questions may help eliminate the possibility of infection with P. falciparum or P. vivax, usually the only two species that can cause severe disease and, in the case of P. falciparum, can rapidly lead to death.

Conventional Microscopy

Often, when the diagnosis of malaria is considered, only a single blood specimen is submitted to the laboratory for examination; however, single films or specimens cannot be relied upon to exclude the diagnosis, especially when partial prophylactic medication or therapy is used. Partial use of antimalarial agents may be responsible for reducing the numbers of organisms in the peripheral blood and lead to a blood smear that contains few organisms and a conclusion that reflects a low parasitemia when in fact serious disease is present. Patients with a relapsing case or an early primary case can also have few organisms in the blood smear. It is recommended that both thick and thin blood films be prepared immediately, and at least 300 oil immersion fields should be examined on both films before a negative report is issued. Since one set of negative smears does not rule out malaria, additional blood specimens should be examined over a 36-h period. Although Giemsa stain has been recommended for all parasitic blood work, the organisms can also be seen if other blood stains, such as Wright’s stain or any of the rapid blood stains, are used. Blood collected with the use of EDTA anticoagulant is preferred over heparin; however, if the blood remains in the tube for approximately an hour or more, true stippling

might not be visible within the infected erythrocytes (e.g., those infected with P. vivax). Using EDTA, if blood is held for more than 2 h prior to blood film preparation, several artifacts may be seen; after 4 to 6 h, some of the parasites will be lost. During the time when the parasites are in the tube of blood, they continue to grow and change according to the life cycle for that species. Also, when using anticoagulants, it is important to remember that the proper ratio between blood and anticoagulant is necessary for good organism morphology—fill the tube with blood. Both thick and thin blood films should be prepared immediately after receipt of the blood. If the specimen is sent to a reference laboratory, both the thick and thin blood films, as well as the tube of blood (room temperature), should be sent. Since this test is always considered a STAT request, it is also important to know what turnaround times are available from the reference laboratory.

If the collecting laboratory performs a STAT dipstick malarial rapid test (Binax NOW Malaria test kit, Inverness Medical/Binax, Scarborough, Maine), the following steps are relevant. The rapid test should be performed immediately on receipt of the blood (STAT). If positive, the result and interpretation should be sent to the physician. If the BinaxNOW is negative, the result should be reported to the physician. HOWEVER, at the same time, the laboratory is responsible for examining and reporting the results of thick and thin blood films (STAT basis), even if the blood is being sent to a reference laboratory.

All requests for malaria diagnosis are considered STAT requests, and specimens should be collected, processed, examined, and reported accordingly. Although other diagnostic tests can be ordered, any request for examination of blood films should include a possible diagnosis of malaria; thus, these requests are always considered STAT. Not only should the blood collection be considered STAT, but also the processing and examination of both thick and thin blood films should be performed immediately on receipt of the blood. Often immunologically naive individuals with no prior exposure to malaria can present to the emergency room or clinic with symptoms such as fever and malaise and a relevant travel history to an area of the world where malaria is endemic. These patients can have very vague symptoms, but they have the potential to become very ill with malaria, even with a low parasitemia (0.0005% to 0.1%).

References

Garcia LS, 2016. Diagnostic Medical Parasitology, 6th Ed. ASM Press, Washington, D.C.

Garcia, LS, 2021. Practical Guide to Diagnostic Parasitology, 3rd Ed. ASM Press, Washington, D.C




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