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A patient is a 52 year-old male from California who has just returned from a trip to Africa. About a month after he returned, he began complaining of fever, headache, and general malaise. The first blood specimens examined using automated instrumentation revealed nothing significant. He continued to have high fevers and severe headaches, and eventually several additional blood specimens were submitted to the microbiology laboratory for examination as thick and thin blood films. The following images were seen - please comment on the possible diagnosis.
1. Blood stain 2. Thick film
3. Blood stain 4. Thick film
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ANSWER AND DISCUSSION OF DIAGNOSTIC QUIZ #58
1. Plasmodium falciparum ring forms (Note appliqué form in upper RBC)
2. Plasmodium falciparum thick blood film (Note: two rings with WBC)
3. Plasmodium malariae developing trophozoite (Note: normal sized RBC and “band” form)
4. Plasmodium malariae thick blood film (Note the developing schizonts)
COMMENTS ON THE PATIENT:
At first glance, the fact that the man had just returned from a trip to Africa might suggest a parasitic infection as a possibility. The patient's symptoms were caused by a mixed infection with Plasmodium falciparum and Plasmodium malariae that he had acquired in Africa. Although the patient had taken prophylaxis for malaria, he had not taken the medication on a regular basis and did not continue to take the medication after returning to the United States. Because he had never had any contact with malaria before (immunologically naive), he began to have symptoms with a very low parasitemia (<0.01%) that was undetectable using automated instrumentation. By the time his symptoms became more severe (several weeks), he had numerous parasites in the peripheral blood. By the time the infection was diagnosed, the man was severely ill.
COMMENTS ON P. FALCIPARUM MALARIA:
P. falciparum tends to invade all ages of RBCs, and the proportion of infected cells may exceed 50%. Schizogony occurs in the internal organs (spleen, liver, bone marrow, etc.) rather than in the circulating blood. Ischemia caused by the plugging of vessels within these organs by masses of parasitized RBCs will produce various symptoms, depending on the organ involved. It has been suggested that a decrease in the ability of the RBCs to change shape when passing through capillaries or the splenic filter may lead to the plugging of the vessels.
Onset of a P. falciparum malaria attack occurs from 8 to 12 days after infection and is preceded by 3 to 4 days of vague symptoms such as aches, pains, headache, fatigue, anorexia, or nausea. The onset is characterized by fever, a more severe headache, and nausea and vomiting, with occasional severe epigastric pain. There may be only a feeling of chilliness at the onset of fever. Periodicity of the cycle will not be established during the early stages, and the presumptive diagnosis may be totally unrelated to a possible malaria infection. If the fever does develop a synchronous cycle, it is usually a cycle of somewhat less than 48 h. True relapses from the liver do not occur, and after a year, recrudescences are rare.
Severe or fatal complications of P. falciparum malaria can occur at any time during the infection and are related to the plugging of vessels in the internal organs, the symptoms depending on the organ(s) involved. The severity of the complications in a malaria infection may not correlate with the parasitemia seen in the peripheral blood, particularly in P. falciparum infections.
Disseminated intravascular coagulation is a rare complication of malaria associated with high parasite burden, pulmonary edema, rapidly developing anemia, and cerebral and renal complications. Vascular endothelial damage from endotoxins and bound parasitized blood cells may lead to clot formation in small vessels. Cerebral malaria is most often seen in P. falciparum malaria, although it can occur in the other types as well. If the onset is gradual, the patient may become disoriented or violent or may develop severe headaches and pass into coma. Some patients, even those who exhibit no prior symptoms, may suddenly become comatose. Physical signs of central nervous system involvement are quite variable, and there is no real correlation between the severity of the symptoms and the peripheral blood parasitemia.
Although malaria is no longer endemic within the United States, this infection is considered to be life threatening, and laboratory requests for blood smear examination and organism identification should be treated as ``STAT'' requests. Malaria is usually associated with patients having a history of travel within an area where malaria is endemic, although other routes of infection are well documented. During 2002 (most recent data available), >45% of the malaria cases reported in the United States were caused by P. falciparum, the most pathogenic of the four species infecting humans.
COMMENTS ON P. MALARIAE MALARIA:
P. malariae invades primarily the older RBCs, so that the number of infected cells is somewhat limited. The incubation period between infection and symptoms may be much longer than that seen with P. vivax or P. ovale malaria, ranging from about 27 to 40 days. Parasites can be found in the bloodstream several days before the initial attack, and the prodromal symptoms may resemble those of P. vivax malaria. A regular periodicity is seen from the beginning, with a more severe paroxysm, including a longer cold stage and more severe symptoms during the hot stage. Collapse during the sweating phase is not uncommon.
Proteinuria is common in P. malariae infections and in children may be associated with clinical signs of the nephrotic syndrome. It has been suggested that kidney problems may result from deposition within the glomeruli of circulating antigen‑antibody complexes in an antigen excess situation seen with a chronic infection. Apparently, the nephrotic syndrome associated with P. malariae infections is unaffected by the administration of steroids. A membranoproliferative type of glomerulonephritis with relatively sparse proliferation of endothelial and mesangial cells is the most common type of lesion seen in quartan malaria. Using immunofluorescence, granular deposits of IgM, IgG, and C3 are seen. Since chronic glomerular disease associated with P. malariae infections is usually not reversible with therapy, genetic and environmental factors may play a role in the nephritic syndrome.
The infection may end with spontaneous recovery, or there may be a recrudescence or series of recrudescences over many years. These patients are left with a latent infection and persisting low‑grade parasitemia for many, many years.
Leukopenia is usually present; however, leukocytosis may be present during the febrile episodes. Concentrations of total plasma proteins are unchanged, although the albumin level may be low and the globulin fraction may be elevated. The increase in the concentration of gamma globulins is caused by the development of antibodies. The level of potassium in serum may also be increased as a result of RBC lysis.
Coincident infection with more than one species of malaria is more common than previously suspected. As an example, in Thailand, up to 30% of patients who present with severe P. falciparum malaria also are infected with P. vivax. Based on overlapping geographic areas where both P. falciparum and P. vivax are found, it is not surprising that dual infections occur. In Africa, dual infections with P. falciparum and P. malariae have also been found. Although the figures may be underestimates, in Gambian children, the prevalence of mixed species varies from <1% to >60%. There has even been reported a rare quadruple malaria infection in a remote area in the western half of New Guinea Island (Irian Jaya Province, Indonesia), and this infection was confirmed using nested PCR and species‑specific primer pairs. In Africa, P. ovale occurs commonly in areas with Duffy‑negative populations who are refractory to P. vivax, thus appearing to prevent the natural coexistence of the two species. However, the Duffy blood group‑negative trait that protects many African populations from P. vivax is considered very rare or nonexistent in New Guinea. Since P. ovale is considered a very unusual finding outside of Africa, the natural occurrence of P. ovale and P. vivax together in the same patient is considered a rare finding.
DIAGNOSIS OF INFECTION:
Frequently, for a number of different reasons, organism recovery and subsequent identification may be 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. When requests for malarial smears are received in the laboratory, some patient history information should be made available to the laboratorian. This information should include the following:
1. Where has the patient been and what was the date of return to the United States? (``Where do you live?'')
2. Has malaria ever been diagnosed in the patient before? If so, what 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 other needle transmission (drug user)?
5. When was the blood drawn, and was the patient symptomatic at the time? Is there any evidence of a fever periodicity? Answers to such questions may help eliminate the possibility of infection with P. falciparum, usually the only species that can rapidly lead to death.
Often the diagnosis of malaria is considered, and 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 within the peripheral blood, thus leading to a blood smear that contains few organisms, which then reflects a low parasitemia when in fact serious disease is present. Patients with a relapse case or an early primary case may also have few organisms in the blood smear.
It is recommended that both thick and thin blood films be prepared upon admission of the patient, and at least 200 to 300 oil immersion fields should be examined on the thin film before a negative report is issued. Since one set of negative films will not rule out malaria, additional blood specimens should be examined over a 36-h time frame. Although Giemsa stain is recommended for all parasitic blood work, the organisms can also be seen with use of other blood stains such as Wright's stain or any of the rapid blood stains. Blood collected with use of EDTA anticoagulant is acceptable; however, if the blood remains in the tube for any length of time, true stippling may not be visible within the infected RBCs (P. vivax, as an example). Also, when using anticoagulants, it is important to remember that the proper ratio between blood and anticoagulant is necessary for good organism morphology. If the blood stands for >2 hrs prior to blood film preparation, organism distortion is very likely, with the morphology beginning to mimic that seen with P. malariae. If the blood stands >6 hrs prior to blood film preparation, organisms will disintegrate.
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 24-h basis, 7 days/week.
Plasmodium falciparum (malignant tertian malaria)
1. 36-48-hour cycle
2. Tends to infect any cell regardless of age, thus very heavy infection may result
3. All sizes of RBCs
4. No Schüffner's dots (Maurer's dots: may be larger, single dots, bluish)
5. Multiple rings/cell (only young rings, gametocytes, and occasional mature schizonts are seen in peripheral blood)
6. Delicate rings, may have two dots of chromatin/ring, appliqué or accolé forms
7. Crescent-shaped gametocytes
NOTE: Without the appliqué form, Schüffner's dots, multiple rings/cell, and other developing stages, differentiation among the species can be very difficult. It is obvious that the early rings of all four species can mimic one another very easily. Remember: One set of negative blood films cannot rule out a malaria infection, and this information must be conveyed to the physician.
Plasmodium malariae (quartan malaria)
1. 72-hour cycle (long incubation period)
2. Tends to infect old cells
3. Normal size RBCs
4. No stippling
5. Thick ring, large nucleus
6. Trophozoite tends to form "bands" across the cell
7. Mature schizont contains 6-12 merozoites (rosette formation)
COMMENTS ON REPORTING PARASITEMIA:
It is important to report the level of parasitemia when blood films are reviewed and found to be positive for malaria parasites. Because of the potential for drug resistance in some of the Plasmodium species, particularly P. falciparum, it is important that every positive smear be assessed and the parasitemia reported using the exact, same method on followup specimens as on the initial specimen. This allows the parasitemia to be followed after therapy has been initiated. In cases where the patient is hospitalized, monitoring should be performed at 24, 48, and 72 hours after initiating therapy. Generally, the parasitemia will drop very quickly within the first 24 hours; however, in cases of drug resistance, the level may appear to drop, then begin to rise again or the level may not decrease, but actually increase over time.
Clinical Laboratory Standards Institute/National Committee for Clinical Laboratory Standards. 2000. Laboratory diagnosis of blood-borne parasitic diseases. Approved Guideline, M15-A. CLSI/NCCLS, Villanova, PA.
Garcia, LS, 2016. Diagnostic Medical Parasitology, 6th Ed., ASM Press, Washington, DC.
Garcia, LS, 2009. Practical Guide to Diagnostic Parasitology, 2nd Ed., ASM Press, Washington, DC