Chapter 13
Malaria: The Clinical Disease
Colonel O` Neill Barrett, Jr., MC, USA (Ret.), and Colonel Raymond W. Blohm, Jr., MC, USA
CLINICAL MANIFESTATIONS
In Vietnam, the typical symptoms of malaria did not differ greatly from classic descriptions of them. The incubation period--the interval from mosquito bite to first symptoms-averages approximately 2 weeks for vivax and ovale infections and can be as short as 9 days for falciparum and as long as 5 weeks for malariae (Belding 1965, pp. 291-96). In American troops taking the combined C-P (chloroquine-primaquine) tablet once weekly the onset of symptoms, as determined by observations from "search and destroy operations," was often delayed as long as 19 days (Blount 1966). Table 44 is a composite of the symptoms and physical findings in 621 cases of malaria acquired in Vietnam. Species differentiation has not been attempted since there are no accurate data providing for a distinction in uncomplicated malaria.
One of the most characteristic features of malaria is periodicity, with fever and chills occurring every second or third day. However, this "typical" picture is not usually present early in the course of disease, when most military patients were seen. Synchronization of parasitic cycles is often established late or not at all during the primary attack (Heineman 1972), especially in the nonimmune subject. Synchronization of fever spikes at the onset of symptoms usually implies relapse (Barrett and Reiley 1971). The typical fever pattern may also be obscured by previous prophylaxis. The nondiagnostic fever curve is especially characteristic of falciparum malaria (Heineman 1972).
The initial fever characteristics of acute malaria may offer some diagnostic clues, however. The temperature nearly always spikes from normal to 103 0 F or higher and returns to normal within 24 hours; it is rarely sustained at a high level (Barrett and Reiley 1971, p. 29). Fever and chills are noted in almost all initial attacks. In second, third, or further attacks, the spiking temperature and shaking chills--and other symptoms in general-become less marked and even less typical; some patients may experience no fever and complain only of malaise.
Headache is present in 75 to 100 percent of the cases but is usually not severe. Persistent or worsening headache may be an early manifestation of cerebral malaria (Daroff et al. 1967). Gastrointestinal symptoms, especially
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nausea and vomiting, occur in about half of malaria cases but are not usually serious; however, they may prevent effective oral therapy. When diarrhea occurs concomitantly, as it did in 35 to 38 percent of cases in two studies, the disease may be mistaken for "gastroenteritis" (Bartelloni, Sheehy, and Tigertt 1967; Quint 1966). Arthralgia and myalgia, especially in the lumbar area, may also be prominent symptoms. Radiologists at the 12th Station Hospital in the Pacific in World War II recognized that the combination of severe back pain and a negative X-ray of the lumbar spine was a frequent manifestation of the disease (MD-R, p. 669). ???
The absence of certain symptoms of common febrile diseases may also facilitate the diagnosis of malaria. Rhinorrhea and nasal congestion are not features of the disease. Cough, if present, occurs only during the febrile period and is rarely productive (Barrett and Reiley 1971).
The physical examination in uncomplicated malaria is not generally helpful. The spleen is palpable in 30 to 40 percent of cases but is not greatly enlarged (Barrett and Reiley 1971; Bartelloni, Sheehy, and Tigertt 1967; McCabe 1966; Deller and Russell 1967). This is in contrast to findings in Vietnamese patients with chronic malaria, among whom the incidence of splenomegaly is 70 to 90 percent (in children and adolescents) and spleens are frequently massively enlarged
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(Colwell, Legters, and Fife 1970). Hepatomegaly occurs in approximately 25 percent of the cases (see table 44) but is usually minimal. The liver is almost universally involved (Deller et al. 1967). Glasser (1967) was the first to call attention to the relative bradycardia which occurs during temperature spikes, especially in cases of P. falciparum infection. Others have subsequently confirmed this observation (Fisher et al. 1970). ???
An evaluation of routine blood counts reveals that patients may have anemia, thrombocytopenia, leukopenia, or any combination of these findings, as a consequence of either malaria or malaria chemotherapy, or of both (Heineman 1972; Sheehy and Reba 1967).
Widely varying rates have been reported for the occurrence of anemia, depending on the definition, the length of illness, and the percentage of falciparum infections in the group (Heineman 1972). In one study of 50 patients, 10 of whom had falciparum malaria, 46 percent (23 patients) had hemoglobin concentrations of less than 12 g per 100 ml (Khan, Zinneman, and Hall 1970). In another study involving only falciparum infections, 29 of 50 patients (58 percent) had hematocrit values of less than 38 percent (Glor 1969). The cause of anemia in these cases is not always clear.
Hemolysis is an integral feature of all malarias and is always present with clinical disease (Conrad 1969). Fulminant hemolysis can be produced by falciparum infections, which attack erythrocytes of all ages. Other forms of human malaria are less virulent because they parasitize red cells of specific ages. The parasites of P. vivax infect reticulocytes while the parasites of P. malariae invade only mature erythrocytes; in these infections, therefore, hemolysis is self limited (Young 1966, p.322).
Relatively few recent studies have been done on red cell abnormalities in malaria, despite the apparent importance of hemolysis in this disease. An immune response has been implicated as the cause of the hemolysis by most investigators, who support this hypothesis with the fact that specific malarial antibodies can be found in convalescent plasma (Zuckerman 1964; McGregor, Carrington, and Cohen 1963). Yet there is little evidence that such antibodies attach to erythrocytes and bring about their premature destruction. Very few malaria patients exhibit a positive antiglobulin (Coombs`) test, and animal studies have not demonstrated either that antibodies adsorb to red cell surfaces or that they affect the lifespan of circulating erythrocytes (Adner, Altstatt, and Conrad 1968; George et al. 1966).
Furthermore, a greater degree of hemolysis occurs than might be expected from the number of parasitized cells in the circulating blood (Zuckerman 1960). Conrad and Dennis (1968) found that the spleen removes parasites from infected erythrocytes and returns to the circulation these injured cells whose survival is shortened. Based on their observations on the increased severity of disease in splenectomized animals and on the potential activation of latent malaria in humans, The Surgeon General in May 1968 directed that no individual whose spleen had been surgically removed or was congenitally absent would be assigned to Vietnam or Thailand (OTSG-MS).
Conrad (1969) postulated that hemolysis is caused by loss of negative charge
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from the surface of red blood cells because the parasite usurps essential metabolic functions of the infected cells.
Intrinsic enzyme abnormalities of the red blood cells found in certain ethnic groups also contributed to hemolysis in some anemic malaria patients on drug therapy. The most common deficiency, involving the enzyme G6 PD (glucose-6-phosphate dehydrogenase) is commonly found in blacks and people of Mediterranean origin and is genetically transmitted as a sex-linked trait. It is fully expressed in males carrying a single dose of the abnormal gene (hemizygote) and in females carrying a double dose of the gene (homozygote) and has intermediate expression in heterozygote females (Tarlov et al. 1962). In the Mediterranean type, the enzyme is almost completely absent; in blacks, sufficient enzyme is present in younger red cells so that only older cells are destroyed, usually resulting in mild, self-limited hemolysis and anemia (Motulsky and Stamatoyannopoulos 1966).
Both primaquine and dapsone produce hemolysis in the WD-deficient individual (Tarlov et al. 1962; Ognibene 1970). Theoretically, at least, all soldiers in Vietnam received primaquine on a weekly basis in the combined C-P tablet. Dapsone prophylaxis was authorized for use by commanders, upon advice of the command surgeon, in hyperendemic areas (USARV Reg); there are no data on the number of troops who received this drug.
There was a small but continuous evacuation of WD-deficient troops from Vietnam because of severe hemolysis secondary to primaquine sensitivity, averaging 17 per month.* Most of these patients were black, although hemolysis supposedly took a mild form in this ethnic group. Despite a recommendation by the USARV (U.S. Army, Vietnam) medical consultant that all individuals, especially blacks, scheduled for Vietnam duty be challenged with C-P before departure from the continental United States,** no official screening policy was adopted.
Another recognized, though uncommon, and preventable cause of anemia in malaria patients was treatment with pyrimethamine, which became a standard component of therapy for falciparum disease in 1967 (OTSG-G). Pyrimethamine acts as a dihydrofolic acid reductase inhibitor, preventing conversion of folic to folinic acid, thus inhibiting nucleic acid synthesis (Kaufman and Geisler 1960). Sheehy and Reba (1967) observed three cases of megaloblastic anemia among 135 patients who received pyrimethamine-quinine treatment for chloroquine resistant falciparum infection. Tong et al. (1970) demonstrated that the incidence of anemia secondary to pyrimethamine treatment, which was 24 percent in their group, could be reduced to 4 percent by the concurrent administration of folic acid, without loss of therapeutic efficacy.
Canfield (1969), in ferrokinetic studies of patients treated with quinine, pyrimethamine, and sulforthodimethoxine (Fanasil), demonstrated delayed red blood cell incorporation of iron until the malaria infection was brought under control.
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*Lt. Col. Andre J. Ognibene, MC, USARV Medical Consultant, 1969: Unpublished data.
** Lt. Col. Andre J. Ognibene, MC, USARV Medical Consultant, 1969: Personal communication.
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Another interesting abnormality of erythrocyte function was reported by Cohen et al. (1968), who studied six soldiers evacuated from Vietnam because of cyanosis which developed while taking antimalarial drugs. The red blood cells of these patients and two other subjects were found to have a markedly decreased concentration of NAD (nicotinamide-adenine dinucleotide) methemoglobin reductase and a decreased capacity to reduce methemoglobin to hemoglobin in vitro. It was demonstrated that chloroquine, primaquine, and dapsone, in doses that have no effect on normal persons, each provoke methemoglobinemia in enzyme-deficient subjects. With greater use of these drugs in combination and with increased troop strength, the problem of cyanosis became more common.
Despite the availability of reasonably detailed data about red blood cells in malaria patients, the leukocyte response in acute malaria received only casual mention in the literature. Clyde (1964) discussed the leukocyte response in an East African population, but his results were obscured by the fact that his studies were performed on individuals partially immune to malaria and among whom there was a high incidence of associated disease. Martelo, Smoller, and Saladin (1969) found leukopenia in 12 percent of 176 individuals who developed malaria following return from Vietnam and leukocytosis in 5 percent. The disease was caused by P. vivax in 93 percent of the group. Goldstein (1968) reported data in a similar group; 30 percent of his 64 patients with falciparum disease and 24 percent of 17 with vivax manifested leukopenia. Fisher et al. (1970) noted these trends but also observed a significant difference in the leukocyte count between races: they found leukopenia in 22 percent of white subjects but in only 12 percent of blacks.
Reiley and Barrett (1971) reported the only detailed study of the leukocyte response in acute malaria. Their series included a total of 404 cases from three separate sources. In one group, 43 cases from the Panama Canal Zone were all caused by P. vivax. The second group, 81 cases treated at the 8th Field Hospital in Nha Trang, was caused by P. falciparum. The third group, 280 cases treated at Tripler General Hospital in Hawaii, occurred in individuals recently returned from Vietnam: 110 were caused by P. falciparum, 159 by P. vivax, and 11 by mixed infection. The data from these three groups were especially valuable since they came from a large number of cases of acute disease in otherwise healthy, nonimmune individuals. Of greatest importance, perhaps, was that they provided additional comparison between vivax and falciparum disease and that the Panama Canal group, which had not received chemoprophylaxis, served as a control in terms of drug effect. Without this control group, the data from Vietnam could have been clouded by the fact that most individuals received weekly C-P tablets and many also received dapsone.
Leukopenia was the most consistent feature of the leukocyte response in this study, occurring in approximately one-third of the cases. There was no difference in the total white count based on patient source. These data are shown in table 45. Chart 16 shows a distribution of leukocyte count in 404 cases by infecting species and, again, no difference is noted. Furthermore, using the Panama Canal cases as a control group, it was shown that leukopenia was not aggravated by the chemoprophylaxis program in Vietnam since white counts were similar in
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TABLE 45.- Distribution of leukocyte count, by patient source, in 404 cases of malaria
both groups. In addition to the decrease in the total leukocyte count, a "left shift" or increase in immature neutrophilic leukocytes was also observed. This finding, present in one-third of the cases, was not uncommon even in patients with leukopenia. Severity of disease, duration of symptoms, and response to therapy were apparently not related to the changes in the neutrophilic leukocyte count.
Eosinophilia was not part of the leukocyte picture in untreated malaria but developed in 30 percent of the patients following treatment with antimalarial drugs. Contrary to older reports in the literature, the monocyte count did not show a significant elevation in most cases although occasionally a striking elevation of the absolute monocyte count was observed.
Despite the potential for the development of the leukopenia secondary to drug treatment, only a few serious problems were reported from medical facilities in Vietnam. In one study at the 6th Convalescent Center, Rogoway (1967) reported severe neutropenia with an absolute granulocyte count less than 400 per mm3 in 10 of 2,200 patients treated between 1 December 1966 and 15 April 1967. Results of bone marrow examination in these patients were compatible with maturation arrest of the myeloid elements. All patients had received a combination of quinine, pyrimethamine, and dapsone, any or all of which can produce leukopenia. Specific drug cause was not established since none of the patients was rechallenged with the drugs.
Dapsone seemed least likely to have been the offending agent in this group since all patients had been on dapsone prophylaxis before the development of malaria. On the other hand, Ognibene (1970) reported the development of agranulocytosis in 16 soldiers in Vietnam who had received daily dapsone prophylaxis against falciparum malaria for 3 weeks to 3 months. There were eight deaths in this group, and this evidence of serious though uncommon toxicity resulted in a limitation in the use of dapsone in U.S. troops.
Thrombocytopenia was reported in 30 percent of the 357 cases summarized by Heineman (1972). Again the mechanism is unclear, although Neva and associates (1970) made a thorough study and drew tentative conclusions. They considered a number of possible mechanisms but rejected them because of lack of evidence in both animal models and human studies. These included the decreased production of platelets by marrow megakaryocytes, hypertrophy of the
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CHART 16.- Distribution of total leukocyte count in 404 cases of malaria
reticuloendothelial system, action of cross-reacting antibodies, and endotoxin mediated reactions. They also excluded consumption coagulopathy or intravascular coagulation since there was no evidence of abnormality or fluctuation of clotting factors or of fibrinolysis. Because of the temporal relationship to the decrease in platelet count, the development of antimalarial antibodies, and a drop in serum complement levels, they proposed that an antigen-antibody complex produces an immunologically mediated thrombocytopenia.
Convincing evidence for DIC (disseminated intravascular coagulation) as a serious complication of malaria in Vietnam is not available, largely because even
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screening studies for this disorder could not be performed due to lack of facilities. In an evaluation of 42 patients with acute renal insufficiency secondary tofalciparum malaria, 7 of 11 patients tested were judged to have evidence of DIC. The only coagulation studies performed, however, were platelet count, PT (prothrombin time), PTT (partialthromboplastin time), and plasma fibrinogen levels (Stone, Hanchett, and Knepshield 1972).Fletcher et al. (1972), studying 26 patients admitted to the Naval Support Activity Station Hospital in Da Nang, measured the PT, PTT, fibrinogen level, platelets, and fibrin split products. Only one patient in this group showed evidence of probable DIC.
Dennis et al. (1967) did coagulation studies of 31 American soldiers evacuated from Vietnam with relapsed chloroquine-resistant falciparum malaria. They found multiple coagulation defects in these patients, including platelet counts below 150,000 per mm3 in two-thirds, and either prolonged prothrombin time or prolonged partial thromboplastin time, or both, in all patients. Depletion of factors V, VII, VIII, and X was shown in specific factor assays. Ten patients had fibrinogen concentrations of less than 200 mg per 100 ml. Heparin therapy, administered to several patients, was followed by an increase in the platelet count and improvement in first and second stage coagulation defects. Recurrence of thrombocytopenia and depression of coagulation factors were noted when the therapy was stopped. In animal studies evaluating the usefulness of heparin therapy in the treatment of malaria, Dennis and Conrad (1968) found that the drug has an antimalarial as well as an anticoagulant effect. This finding has not yet been confirmed in man, however.
Cerebral malaria was recognized as a complication of falciparum infection in Vietnam. The diagnosis, often an imprecise one, was made on the basis of confirmed parasitemia and neurologic manifestations which could not be explained on the basis of hyperpyrexia or associated metabolic abnormalities. The incidence in Vietnam was estimated to be 1.6 percent (of 2,600 total malaria cases). This figure is remarkably similar to that reported in World War II, which was 2.3 percent (of 6,059 total cases) for U.S. and Allied troops (Daroff et al. 1967; Carr 1967).
The symptoms of cerebral malaria are frequently nonspecific, especially since the functional versatility of the brain permits expression of a number of syndromes. The best data for Vietnam are from Daroff et al. (1967) and Carr (1967). Based on 19 cases studied, Carr tabulated the following symptoms of cerebral malaria:
Headache - 17 Convulsions - 9
Disorientation - 16 Restless agitation - 7
Recent memory loss - 16 Abnormal platar refles - 6
Hyperreflexia - 13 Pseudobulbar affect - 6
Ankle clonus - 12 Papillema - 4
Coma - 11 Decerebrate rigidity - 1
Ataxiz - 10
Daroff and associates classified the 19 cases by five syndromes characterize cerebral malaria, as follows:
Disturbed consciousness - 8 Chorea or myoclonus - 3
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Organic mental syndrome - 4 Focal disorders - 1
Personality changes - 3
In the classic form, coma develops, usually insidiously but at times with alarming rapidity. The comatose state is unusual in that the patient may appear to be awake with eyes open but roving, simulating the postictal state or pseudobulbar palsy.
The pathophysiology of cerebral malaria is not well understood but has been attributed to stickiness of parasitized erythrocytes with distention and plugging of small cerebral capillaries (Spitz 1946). Pathologic correlation with cases of cerebral malaria in Vietnam is difficult to obtain. None of the 19 patients studied by Daroff et al. and Carr died. In autopsy data from the case cited by Kiel (1968), there was gross evidence of cerebral edema and infarction of the pituitary gland. No microscopic material was described.
Renal involvement is common, especially in falciparum malaria. The term "blackwater fever" has sometimes been confused with renal failure but, in fact, simply represents a urinary manifestation of rapid or severe intravascular hemolysis. It does not imply a renal function abnormality; rather, it indicates that the plasma hemoglobin-binding concentration of haptoglobin has been exceeded and that the kidney is excreting hemoglobin or its breakdown products (Neva et al. 1970). Renal failure may occur during infection with P. falciparum, however, associated sometimes with hemoglobinuria and frequently with fluid and electrolyte abnormalities, hypotension, and DIC (Canfield 1969).
Sheehy and Reba (1967) reported on 14 patients in Vietnam who developed acute renal insufficiency secondary to falciparum disease in 1965. They were evacuated to Clark Air Force Base in the Philippines because there were no facilities for hemodialysis in Vietnam at that time. The mortality rate was 50 percent in this group: four patients died before definitive treatment could be started, and three died of coexistent complications. This prompted the decision to establish a renal team in Vietnam capable of dealing with the problems of acute renal insufficiency. The history of the team and the results obtained in the treatment of this disorder are described in detail in chapters 20 and 21 of this text.
The frequency and extent of renal lesions in patients not manifesting overt renal failure have not been determined. However, proteinuria has been noted in about one-half of patients with falciparum disease, and azotemia is seen in approximately 7.8 percent (Heineman 1972). Ward and Kibukamusoke (1969) have presented evidence indicating that the glomerular lesion is an immunologic disorder characterized by deposits of soluble immune complexes.
Respiratory symptoms and signs occur in 3 to 10 percent of patients with acute falciparum malaria (Applebaum and Shrager 1944; Bergin 1967; Neva et al. 1970, p. 304), ranging from mild upper respiratory complaints to fatal pulmonary edema. Applebaum and Shrager described the occurrence of clinical pneumonia in 3.7 percent of their group of 87 military patients in Panama. Based on the therapeutic response, they classified the disease as bacterial pneumonia with a satisfactory response to sulfonamides, atypical (viral) pneumonia with inadequate response to therapy, or malarial involvement with response to an-
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timalarial drugs. Spitz (1946) studied 50 cases of fatal falciparum malaria at the Armed Forces Institute of Pathology and found evidence of bronchial pneumonia in 28 percent and interstitial pneumonia in 12 percent.
Clinical pneumonia was not a prominent feature of malaria in Vietnam (Heineman 1972).Fletcher et al. (1972) performed pulmonary function studies and arterial blood gas determinations in 26 randomly selected patients with acute falciparummalaria in Da Nang. All had previously been in good health and had undergone no known treatment for malaria. One patient had an initial arterial oxygen tension of 57 mm of mercury. He had recently recovered from an upper respiratory infection but was asymptomatic from a pulmonary standpoint. There was no clinical or radiographic evidence of pulmonary disease. All values in this patient returned to normal within 24 hours and remained so throughout hospitalization. Pulmonary studies on the other 25 patients were normal on admission and remained so during the study.
Acute pulmonary edema is an uncommon but serious complication of falciparum malaria, occurring in less than 1 percent of all cases (Fletcher et al. 1972). Newman and Hale (1969) reported six cases of this complication in a series of 1,200 patients treated for malaria at the 71st Evacuation Hospital in Vietnam. There were two deaths in this group. Of six patients who died in Hagan`s (1970) series of 1,146 cases in Vietnam in 1969, three had significant pulmonary involvement.
Although acute pulmonary edema was first described as a complication of malaria in 1905 and has often been observed at autopsy, its pathogenesis has not been completely examined (Brooks et al. 1968). In some cases, it may result from fluid overload and coexisting renal failure. Sheehy and Reba (1967), for example, described progressive weight gain, dyspnea, tachycardia, rales, and pitting edema in some cases. Stone, Hanchett, and Knepshield (1972) described pulmonary edema in 8 of 12 deaths in their series of 42 patients with malaria and acute renal insufficiency.
Brooks et al. (1968) performed detailed clinical and pathological studies on five patients with falciparum disease who died of acute pulmonary edema and found that development of the complication could not be related to fluid retention, cardiac decompensation, or peripheral circulatory collapse. Central venous pressure remained normal throughout the course of the edema in the three patients in which it was monitored. None of these patients had roentgenographic evidence of cardiac enlargement. On the other hand, all patients had signs of central nervous system dysfunction, and the onset of the edema correlated closely with the degree of cerebral depression. On the basis of the pathologic and physiologic studies, Brooks and associates proposed the following explanation of the pathogenesis. Generalized vasodilatation, with clinical manifestations of a decreased effective circulating volume, occurs during the acute phase of falciparum malaria, accompanied by a decrease in organ perfusion. Further aggravation is produced by local changes in the microcirculation, aggregation of erythrocytes, diminished flow, increased capillary permeability, and interstitial edema. This hypothesis is supported by the autopsy results. Severe congestion of pulmonary capillaries, thickened alveolar septums, diffuse pulmonary edema,
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focal hyaline membrane formation, and scattered areas of intra-alveolar hemorrhage were among the major microscopic findings. No significant cardiac abnormalities were observed.
This picture, therefore, closely resembles "shock lung" or the "adult respiratory distress syndrome" and is probably caused primarily by abnormalities of the pulmonary microcirculation (Heineman 1972, p.612; Brooks et al. 1968). Treatment for this complication involves the usual modalities for pulmonary edema, including meticulous attention to maintaining pO2 (Neva et al. 1970, p.304). In addition, there is evidence that large doses of adrenal corticosteroids may help bring about reversal of the process. Newman and Hale (1969) thought that methylprednisolone sodium succinate enhanced recovery in the four survivors among their six patients, and Bergin (1967) reported a similar observation.
There have been few clinical studies concerning liver involvement in malaria in Vietnam, although the liver appears to be almost universally involved; most observations have dealt with events that occurred during the early and late exoerythrocytic phase in experimental malaria. Generally malarial hepatitis is of no clinical significance (Heineman 1972, p.613). Deller et al. (1967) reported one of the few extensive clinical evaluations of it, in a study of 38 patients admitted to the 93d Evacuation Hospital with acute falciparum malaria during a 2-month period in 1966. Liver function studies were performed daily for 3 days. Enzyme abnormalities were detected in approximately two-thirds of the group and some histologic abnormality was discovered by liver biopsy in all. Histologic changes included Kupffer`s cell hyperplasia, increased pigmentation, periportal mononuclear cell infiltration, and increased hepatocyte activity. De Brito, Barone, and Faria (1969) had similar findings.
DIAGNOSIS
The diagnosis of malaria depends primarily upon identifying the parasite in a peripheral blood smear (Neva et al. 1970, p. 296). As noted earlier, the symptoms and signs lack the specificity required for diagnosis except perhaps in the unusual case in which a true periodic fever is present. Once malaria is suspected on the basis of history, including travel through an endemic area and a compatible clinical syndrome, laboratory confirmation is necessary (Heineman 1972, p. 613). Standard peripheral blood smears stained with Wright`s stain were used to diagnose the early cases of malaria among troops in Vietnam. Diagnosis was made by identification of a characteristic trophozoite or schizont within erythrocytes. This technique was quite effective but time-consuming, and evaluation of several slides was frequently necessary; most clinicians required three negative malaria preps before discarding the diagnosis in a patient with an unexplained febrile illness (Kiel 1968, pp.13-14).
Blohm * made an interesting observation which reflected the inexperience of most American physicians with the disease. He noted that the transient ap-
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* Col. Raymond W. Blohm, MC: Personal communication.
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pearance of gametocytes in the peripheral blood 6 to 12 days after treatment for falciparum malaria was not recognized as part of the natural history of the disease. Thus, patients were unnecessarily retreated for incorrectly diagnosed recurrence until this information was disseminated to treatment facilities.?
Because of the increasing volume of work, the thin smear became impractical for routine use in most laboratories in Vietnam, and the thick smear, from which the diagnosis in malaria is most easily made, was "rediscovered." It was used primarily as a screening technique; if it was negative, the thin smear was not examined; if it was positive, the thin smear was studied to determine specific species and parasite index. The parasite index was categorized as mild, moderate, marked, or severe. Mild infection meant less than 9 trophozoites per oil immersion field on thick smear and moderate infection meant 10 to 99. If more than 30 were counted, the thin smear was examined to exclude marked infection, defined as 1- to 9-percent infestation with trophozoites on thin smear. Severe infection was defined as greater than 10-percent trophozoite parasitemia on thin smear (Kiel 1968, p. 14).
The diagnosis of vivax infection was made when large ragged trophozoites were found in enlarged red cells. For falciparum malaria, diagnostic criteria included: small trophozoites in normal-sized erythrocytes, double chromatin dots, multiple trophozoites in the same cell, applique forms, and characteristic gametocytes (Kiel 1968, p.15; Erickson 1967). Price, at the Armed Forces Institute of Pathology, called attention to several other characteristics of falciparum infection (Kiel 1968, pp.15-16). "Teardrop" forms are small trophozoites which lack a prominent vacuole, common in patients who have had recurrent attacks of malaria with long remissions in between. "Flag" forms are parasites having large rings of thickened cytoplasm at one point, resembling a "flag on its string." "Tenue" forms show very fine ameboid cytoplasm with small and often multiple chromatin dots. Patients in whom this form persists may be seriously ill yet show a relatively low parasitemia. "Bird`s-eye" forms show cytoplasm completely surrounding the nucleus, which appears as a free-standing strip or a dot.
In addition to the standard thick smear concentration technique, several other diagnostic techniques have been used, with varying degrees of success. Keffer (1966) described concentration with saponin hemolysis of erythrocytes. Cranmore, at the 406th Mobile Medical Laboratory in Saigon, devised a venous blood formaldehyde concentration technique (Kiel 1968, p.16), and Van den Berghe and Chardome (1951) employed scarification of skin in the scapula area to increase the ratio of parasites to red blood cells.
The diagnosis of malaria in American troops in Vietnam was not always easy. Erickson (1967) reviewed the problem, emphasizing the frequency and sources of errors, and made recommendations to laboratory officers and technicians. His study included an examination of the first 300 slides from patients referred to the 6th Convalescent Center at Cam Ranh Bay. Very few of the patients had had thick smears made and even when they had, they were of little value because cells had not been properly lysed. Most of the slides had been prepared with Wright`s stain. They were generally overstained and contained
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numerous artifacts. Use of improperly washed slides resulted in clumping of red blood cells on thin smeared preparations. Improperly prepared buffer contained free-living protozoa which led to erroneous diagnoses because of their resemblance to malaria parasites. False positive diagnoses were made from 42 (14 percent) of the 300 slides. Incorrect species identification was made from 14 (4.7 percent) and infections by more than one species in the same patient were missed in 15 other cases; a frequent error was failure to recognize falciparum infection on a slide known to contain vivax organisms. The results of this study led to development of the standardized diagnostic techniques subsequently used in Vietnam.
Fulkerson (1970) performed an interesting but unrewarding study attempting to hasten the appearance of trophozoites in the treated patient liable to relapse. He developed an "Exercise Provocative Test," based on the clinical observation that relapse of falciparum disease tended to occur shortly after strenuous physical activity. In essence, 22 patients who had recently been treated for falciparum malaria exercised by jogging 1 mile in 12 minutes. Malaria smears were obtained immediately before and after jogging, and 1, 3, 12, and 24 hours afterward. Any smear in which trophozoites appeared within 24 hours was considered positive. None of the patients in the study had a positive response, however. Only one had to terminate the study early because of fatigue.
The occurrence of immunity in malaria is well known. The progressive mildness of relapses, eventual spontaneous cure, and survival of indigenous populations in endemic areas are attributed to this phenomenon (Heineman 1972, p.6). As early as 1956, a constant relationship between the rising concentration of serum gamma globulin and the acquisition of clinical immunity to plasmodial infection was demonstrated, in field studies in Africa (McGregor et al. 1956). While a humoral component for such immunity was suspected, there was no practical method by which antibody could be specifically measured and related to alterations in gamma globulin levels. Furthermore, alterations in gamma globulin levels were difficult to interpret in subjects living in hyperendemic areas since preinfection levels were not known (Tobie et al. 1966).
However, the study of the primary malaria attack in human volunteers and the use of specific immunologic techniques has produced data concerning preinfection levels of immunoglobulins and alterations following infection. In a study from the National Institutes of Health (Tobie et al. 1966), immunoglobulins IgM, IgG, and IgA were quantitated in 12 volunteers infected with P. vivax and 5 with P. cynomolgi. Each subject`s serum levels were measured before, during, and after the primary malarial attack and in one case after relapse. All subjects synthesized large amounts of IgM globulin. The volunteer with vivax who relapsed produced essentially as much IgM during the secondary response as he did initially. All volunteers showed large increases in absolute values for IgG, but the percentages of increase were less striking in IgG and IgA than in the macroglobulins. Similar data on persons infected with P. falciparum are not yet available. These immunologic techniques have also allowed more precise epidemiological data to be collected, and current evidence indicates that circulating antibody is of both protective and diagnostic value (Heineman 1972; Kagan, Matthews, and Sulzer 1969).
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Two basic immunologic techniques have been developed, each with subsequent modifications (Kagan, Matthews, and Sulzer 1969). Desowitz and Stein (1962; Stein and Desowitz 1964) developed an IHA (indirect hemagglutination) test utilizing formalin and sheep red cells treated with tannic acid and sensitized with antigens from P. cynomolgi and P. coatneyi. Mahoney, Redington, and Schoenbechler (1966) developed a more sensitive and specific test by systematically extracting antigens from crude P. knowlesi and P. falciparum suspensions. Subsequently, Rogers, Fried, and Kagan (1968) described a microhemagglutination test with antigen from P. knowlesi using methods similar to Mahoney`s. Antigen preparations used in the IHA test had been prepared by lysis of parasitized cells followed by extraction of antigens from the "freed" parasites, the initial lysate from the infected cells being discarded. Wellde et al. (1969) developed a micro method using lysates of parasitized erythrocytes as antigen. This technique has proved to be highly sensitive and requires only small amounts of both antigen and serums, making it a practical tool for seroepidemiological studies. D`Antonio, Von Doenhoff, and Fife (1966) also described a technique of antigen purification using the principle of preferential fragmentation by controlled pressure in a French press which was free of erythrocyte contaminants.
Several uses of the IHA techniques have been suggested, including delineation of the extent of malarial transmission, detection of focal outbreaks in an endemic area, monitoring seasonal changes in malaria transmission, and assess ing the efficacy of chemoprophylaxis and eradication programs (Kagan, Matthews, and Sulzer 1969, pp. 1033-36). Recent outbreaks of malaria can be detected only by testing young people or by noting a rise in the geometric mean titer over a given period of time because of the long duration of the antibody in infected persons (Collins, Skinner, and Jeffery 1968).?
The IFA (indirect fluorescent antibody) test for malaria was introduced initially by Tobie and Coatney (1961) and later modified by Kagan, Matthews, and Sulzer (1969). It is now the most widely used serologic technique for the diagnosis of malaria, although it is not practical for mass screening because of technical difficulties. The sensitivity and specificity of the tests are very high, specificity being greater than 99 percent (Sulzer, Wilson, and Hall 1969). The IFA test is especially valuable in determining the plasmodium species when a determination cannot be made with stained slides. Etiologic diagnosis may be difficult because of distortion of parasites by drugs, improper preparation of slides, or scanty parasitemia. The method may also be used to detect responsible donors in transfusion-induced malaria or to screen high-risk potential donors (Kagan, Matthews, and Sulzer 1969, p. 1039).
The rise and fall of antibody titer in returning military personnel may be useful diagnostically in that high antibody titer may indicate recent or current infection, especially if there is no history of recent treatment (Wilson, Sulzer, and Runcik 1970). Leibovitz et al. (1969) reported significant titers in 49 (26 percent) of 183 returning Vietnam servicemen, none of whom had past or present symptoms of clinical malaria.?
Sadun et al. (1969) performed studies on human volunteers infected with
309
either P. falciparum or P. vivax to determine and compare the time course of development of fluorescent and hemagglutinating antibodies. Both tests were equally sensitive and specific for following the course of antibody development in either falciparum or vivax malaria. While P. falciparum antibody titers were usually higher and more persistent than P. vivax titers, the antibody curves followed almost parallel lines in the two tests.
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