Mal Aire y La Cruz Maltesa

So why speak about these 2 topics on the same post? Because they’re essentially the same disease; they are parasites that have a predilection for red blood cells and lead to hemolysis and other symptoms. Further, their diagnosis is similar as are their therapeutics. In other words, Babesia is the North American Malaria. Which is interesting, to say the least.

Lifecycle of Malaria

Malaria is caused by the Plasmodium parasite, which belongs to the same group of protozoa as Babesia (not surprisingly), but also Toxoplasma (7). These are transmitted by the Anopheles mosquito. So what happens?

1. Mosquitoes feed into a human. As they do this, they inject sporozoites
2. Sporozoites invade into hepatocytes, which differentiate into schizont
   1. If you are dealing with P. ovale or P. vivax, then these can enter a stage called hepatic schizont. There is no symptoms but they can reactivate
3. Schizont ruptures from hepatocytes, releasing merozoites (anywhere from 10k to 30k merozoites per infected hepatocyte) which can invade uninfected RBCs
   1. Within RBCs, merozoites develop ring forms (trophozoites) and then into schizonts over 48hrs (P. falciparum/P.vivax/P.ovale) or 72hrs (P. malaria).
   1. A subset of these develop into gametocytes and are taken up by anopheles mosquito, starting the cycle anew

Here is a clear diagram from UpToDate. One of the important things about the life cycles is that it has treatment implications (more on this later). Because all other species outside of falciparum can stay in the liver, it can come back and reactivate in patients even after treating the “bloodborne” malaria:

Thick and Thin Smears

This is the gold standard of diagnosis for Malaria and Babesia. This allows you to see, with a light microscopy, the parasites after staining with either Giemsa or Wright’s stains. What is the concept behind this?

• Thin smear: it is exactly as you think it is. You get a drop of blood and spread it with a spreader slide along the width of the slide. The advantage here is that you can see the parasites within the RBCs, and allows you to identify the species
• Thick smear: spread the drop in a circle to the size of a dime. Do not spread too thin. This allows mechanical hemolysis of RBCs, which then allows you to visualize parasites outside of the RBCs. In other words, you are able to “pack a bunch of RBCs” into the film (up to 20-30 times as dense as a regular thin film) which increases sensitivity of a thick smear (1). Indeed, this is a better method for detecting low levels of parasitemia (5), however due to the lysis of RBCs it makes it more difficult to differentiate parasites from WBCs or platelets.

The sensitivity of blood smears has been evaluated in very early studies (1, 2). This, of course, depends on the operator and how long they look at each film. For instance, one early study (2) found that looking at a thin film for 10 minutes by experienced operators had similar results to looking at a thick film for 3 minutes. Further, that same study found that as few as 1 parasite per 1000 fields in thin films and as few as 2 parasites per 300 fields could be found. Doing some fancy math, this gives a threshold of four or more parasites per microliter of blood for detection of parasites in microscopy, though this takes into account an experienced operator, which equals to roughly 0.0001 to 0.0005 percent parasitemia. A survey of thick and thin smears from the UK (6) found that most routine laboratories achieve a n average sensitivity of detection of 0.01% RBC infected, or 500 parasites/uL. Furthermore, when compared to a reference lab, most labs were able to correctly identify the species of malaria 77.1% of the time in single species infections.

In another study, 711 slides collected during epidemiological surveys were re-evaluated. While in general there was good agreement for P. falciparum (kappa index 0.79), there was significant disagreement at low parasite densities, with 53 out of 71 discordant  samples with parasite density <100/uL (in other words, examiners did not agree with regards to the degree of parasitic index). Once low parasite densities were eliminated, the inter-observer agreement was higher (kappa index 0.95). It must be states these sensitives depend on who is doing the smears. Indeed, a survey of 278 centers (3) found that only 5 out of 149 responding centers were in compliance with CLSI criteria for malaria testing.

While this next part is not terribly important from a clinical perspective, it is nevertheless interesting to think about. Given the volume and mechanics of each of the different films, conversion factors have been implemented (2), which take into account the number of leukocytes, RBCs, and parasites that are lost due to staining. They calculate roughly 65% loss of parasites in thick films, with corrected parasite counts in thick films (based on leukocyte numbers, RBCs, and volume) being roughly 2.5 times that of thin films. Further, parasite density was determined by counting the number of trophozoites in 100 oil immersion fields and multiply it by four to give parasites per uL. Another method involves counting the number of parasites until 200 WBCs have been counted (assuming an average of WBC 8000/uL) after which, you can multiply by 40 to give you the number of parasites/uL of blood (7). Parasitemia percentage is then calculated by dividing the parasite density by 4,000,000 (average number of RBC/uL of blood) and multiplying by 100 (7).

Procedure

So I’ve mentioned thick and thin smears above, and how, in experienced hands, they can detect low levels of parasitemia. In general:

• thick and thin films should be done within 24 hours of patient presentation (4). These are typically picked from capillary-rich area such as fingerstick or earlobe, as they have high density of developed trophozoites or schizonts (5)
• If initially negative, repeat smears every 12-24 hours for a total of 3 sets before ruling out malaria
• Estimate parasite density by counting the percentage of infected RBCs after counting anywhere from 500 to 2000 RBCs.

Additionally, you can perform either of the corrections noted above. As an example of the differences in thick and thin films, here are the trophozoites (the “ring” stage), with the upper line looking at thin films and the lower looking at thick films (8):

How do you differentiate between all these malaria species? This is not a question for most of us (unless you need to take ID boards, in which case, good luck), but I figured I could lend some sort of insight. First, we need to figure out the stage:

• Trophozoite – typically a ring-form, with one to two chromatin (the tiny red dots in the middle)
• Schizont – lots of “red dots” representing asexual reproduction (aka division of chromatin into 2). Watch for chromatin bodies and larger cytoplasm

• Gametocyte – weird looking, either oblong or large banana-shaped. Watch for concentration of chromatin on one side of the cytoplasm.

The hard part is telling each of them apart. In general, P. falciparum is the most common type of malaria, which tends to form very nice rings as trophozoites and on thick films, they look very uniform. This is the easiest one to tell apart from the others:

Rapid Malaria Diagnosis

These use immunochromatography to work. Essentially, a patient’s blood is put into the adsorbtion pad, allowing its migration down the sheet. At the beginning, a monoclonal antibody captures the parasitic antigen (more on this later), and it moves down the sheet. As it comes down, it encounters 2 other antibodies, a pan-specific antibody or a P. falciparum specific antibody:

These parasitic antigens come in 2 flavors: the histidine-rich protein-2 (HRP-2), which is present in all P. falciparum parasites and is the first antigen to develop and the P. falciparum-specific lactate dehydrogenase (pLDH), of which some substrates can also differentiate other types of malaria. Early data compared the performance of PfHRP-2 among 3 groups in comparison to microscopy (9). The overall sensitivity was 96.5-100% and specificity was 95% when there were >60 parasites/uL:

A prospective study from France (10) evaluated the NOW ICT (HRP-2 assay) and the OptiMAL assay (pLDH assay) against both RT-PCR and microscopy. The sensitivity for NOW ICT was higher than that for OPTIMAL but both had high specificity in this study:

Another study of 256 patients (11) found that when compared to PCR, the sensitivity of the NOW ICT was 95.5% for detecting pure P. falciparum infections, 94.3% for P. falciparum mixed with other infections, 86.7% for pure P. vivax infection, and 93.5% for non-falciparum infections. Specificity was high at 98.7% When compared to microscopy, the NOW ICT had a sensitivity of 96% for falciparum infections and 84.7% for non-falciparum infections with equally high specificity. In another cohort study from Peru (12), the sensitivity of the OptiMAL assay was found to be 92.3% with specificity of 100% for P. vivax infections, with higher sensitivity the higher the degree of parasitemia:

There are a few issues with these. First of all, HRP-2 based detection is limited to P. falciparum so you cannot use these to differentiate other types of malaria. Further, these tests can be positive up to 28 days, so they’re good for screening but cannot be used to assess therapy. Third, the sensitivity is related to the degree of parasitemia. Despite this, these are fairly good tests given the lack of experience with the gold standard that is microscopy. A review of the different assays are highlighted below (5):

While I did not mention this, PCR is a fairly high sensitive modality that is usually reserved for reference laboratories for research and epidemiological purposes. Part of the reason behind this is the fact it is time consuming, there is high cost of initial equipment set up, and in some cases, the infrastructure may prevent reagents to be obtained. Given the locations of many malaria cases, it is not surprising this is not widely used. However, data suggests it is highly sensitive. For instance, one study (14) found that nested PCR assay was able to detect Plasmodium spp infections down to 0.4 p/Ul, while multi-plex PCR being able to detect P. malareie down to 0.04p uL.

Babesia – Lifecycle

Initially known as “Nantucket Fever,” this disease is caused by intraerythrocytic protozoa and is related to Plasmodium spp. There are several types of species, but I’ll talk about 3. The major one that infects people in the Northeast US (including Minnesota and Wisconsin) is B. microti (13), with other clades including B. duncai and B. dunaci-type organisms that have been identified in Washington State and California. Further, Europe tends to see B. divergens as well. While the Ixodes scapularis is the main mode of transmission of B. microti and Ixodes Ricinus is the vector for B. divergens, the vector for B. dunacani is not known. One thing to note about Ixodes scapularis is the fact it also transmits Lyme disease, Anaplasma, and Powassan virus (15).

Some of the terminology may be a bit confusing, however I’ll try to clarify it as best as possible. For the purposes of this discussion (and at the risk of having legitimate biologist pissed at me), one way of thinking about the stages is as such:

• Eggs – the babies (planted Groot)
• Larva – toddlers (baby Groot from GoT2)
• Nymphs – teens (teen Groot from Infinity War)
• Adults – adult Groot from GoTG

Of course, there are way more complicated differences between larva and nymphs, but for now, this will suffice and let us know how (and why) the lifecycle takes around 2 years to complete. First, adults are not typically infected at the start of the cycle (7). Rather, they feed on the white-tailed deer to give rise to eggs and more ticks. Ticks feed in the fall of year 1, lay eggs in spring of year 2. From here:

• Eggs hatch during the summer of year 2 into larvae.
• In late summer, the larvae feed in smaller rodents (remember, they’re toddlers). Some of these, including the white-footed mouse, are reservoir of Babesia. RBCs with Babesia grow in the midgut
• In spring of year 3, the larvae grow up into nymphs (not exactly what happens, but makes sense in this context). These nymphs are already infected.
• In early summer of year 3, nymphs feed again on rodents but also on humans that tend to get out of school and work during the summer and go hiking. Hence, this is when people get infected. 

Since ticks do not have the organism in the salivary glands, initial feeding does not confer infection. Rather, the organisms have to migrate from the gut into the salivary glands that takes anywhere from 24-48hrs (16). Similar to malaria, once sporozoites get into the bloodstream they invade RBCs and these undergo asexual reproduction.

Thick and Thin Films

Similar to malaria, Babesia is primarily diagnosed by looking at blood films. And similar to malaria, the sensitivity depends on who is looking at the microscope. For instance, sensitivities for films has been quoted to be as low as 1 parasite per 105 – 106 erythrocytes (16) but this depends on the experience of the microscopist. The other issue is that, at certain stages, it actually looks like malaria. The differences are highlighted in this table (17):

In general, babesia RBCs tend to keep their shape (which is why they are not detected in automated systems), they do not have stippling, tend to have more “oddly” shaped ring forms and multiple parasites within the RBC is common.

Other Diagnostic Modalities

The major diagnostic modality is PCR. It has a high sensitivity and specificity. For instance, an aearly study found that real-time DNA PCR was able to detect roughly 100 copies (18) while another was able to detect down to 10 copies in both human blood samples and ticks (19):

Indirect immunofluorescence assay has been used as well. For instance, one study (20) found the sensitivity of IgM for acute infection to be 91% using a cut-off of 1:32 and 89% if using 1:64. Specificity in both instances was high at 99% when comparing to PCR. This was also seen in another study evaluating four laboratories, with range of sensitivity of 88-96% (21). Typically, IgM antibodies are detected in the first 2 weeks. These can rise up to over 1:1024 during the first few weeks, as seen in a study of 16 patients and come down to 1:64 anywhere from 8-12 months later (22). In general, IgG should be followed by convalescent sera to see a four-fold rise in titer to confirm the diagnosis, as IgG can be positive in the absence of symptoms.  

Symptoms

This is a waste basket, to be fair. Symptoms here are very much non-specific, and range from fever, chills, malaise, nausea, vomiting, sweats, and anything in between. If you fancy, here are a few data points for each:

• Malaria (23):

Symptoms	Percentage
Fever	100%
Headache	100%
Weakness	94%
Night Sweats	91%
Insomina	69%
Arthralgias	59%
Myalgias	56%
Diarrhea	13%
Abdominal cramps	8%

• Babesia (15):

In those cases you see in the hospital, you’ll also see some traces of hemolytic anemia, including low haptoglobin, high LDH, anemia, high reticulocyte count. Any symptoms I mention will not be useful clinically, since they can resemble flu, COVID, Staph, etc. The one thing to keep in mind in those who are sick is the travel history. If they have been to the northeast or Latin America/Africa and have a febrile illness after a few weeks, it would be best to send 3 thick and thin smears to complete the work up.

Having said that, there are a few interesting tidbits for each of these

1. Malaria – incubation varies depending on the type of malaria, but typically at around 2 weeks after exposure is when you should expect symptoms.

The classic presentation is the “malaria paroxysms” (24). This comprises 3 stages. It starts with a 15-60 min cold stage (shivering) followed by a hot stage, that lasts 2-6hrs (fevers, reaching up to 41 C) along with flushed skin and headache. This is then followed by a 2-4hr sweating stage, where fever drops and the patient sweats (7,24). The basic thought is these match up with the rupture of schizonts, which as seen above, varies depending on the type of malaria. This only holds true in the later courses. IN the acute course, fevers tend to be random, so if a patient does not have the classic “tertian” or “quartian” fever, do not rule it out:

There are a few symptoms to go over in terms of severe malaria:

• Cerebral malaria – part of this seems to stem from sequestration of infected erythrocyte sin cerebral microvessels. A prospective study of children (25) found that 47.6% of patients with malaria had some sort of CNS involvement. The most common were seizures (37.5%), prostration (20.6%) and impaired consciousness (13.2%). Variables associated with neurological involvement included metabolic acidosis, hypoglycemia, and fever lasting <2 days:

Further, coma and seizures were also associated with higher mortality.

Diagnosis is made by the presence of malaria in films as well CNS abnormalities in the absence of other etiologies. CSF in patients with malaria (26) compared to viral encephalitis and controls were more likely to show low glucose, high ADA, and low white cells:

Certain variables have been used to differentiate cerebral malaria and viral encephalitis, with reasonable sensitivity:

Hypoglycemia – this is likely related to hyperinsulinemia in context of pancreatic islet cell stimulation by parasite-derived factors. This can exacerbate seizures and lead to a coma.

Acidosis – this is usually seen in up to 85% of cases of severe malaria (7). This is likely related due to hypovolemia, sequestration and microvascular obstruction, and anemia, leading to lactic acidosis.

Babesia – incubation is 1-4 weeks if hiking; up to 9 weeks if transfused, with one reported case presenting up to 6 months following transfusion (13)

The big issue here is the immunosuppressed. In general, those who have had splenectomy tend to have severe presentations (either surgical or auto-infarct, as in those with sickle cell). This is because RBCs with parasites are taken up by WBCs in the spleen. Further, TNF plays a role, so drugs that inhibit this also put patients at risk of severe disease. This is the case for B. microti. If you look at guidelines from Europe, you’ll notice that B. divergens tends to be severe.

A retrospective cohort (27) evaluated 139 patients with babesiosis and found that male sex, low WBCs, elevated alkaline phosphatase, and high parasitemia were associated with worse outcomes (death, ICU admission, hospital stay >2 weeks):

In a univariate analysis, splenectomy was also associated with worse outcomes:               

Treatment – Malaria

In general, this falls into one of 2 camps: chloroquine sensitive and chloroquine resistance. By far, if you have a plasmodium that is sensitive to chloroquine, you can use that or HCQ. If, however, you have chloroquine resistance, then therapy is usually a combination of artemisinin and an oral compound. In fact, the WHO guidelines (31) suggest using any combination as first line therapy for P. falciparum. These drugs have broad spectrum activity against all plasmodium species, with artemether, artemotil, and artemisinin being converted into dihydroartemisinin in vivo.

These compounds have a parasite reduction ratios of ~10,000 fold per cycle, reducing the number of parasites in the body by around one hundred million-fold (28). This allows them to reduce the number of parasites in the body to a tiny fraction after about 2 asexual cycles (<0.0001%). However, the drug has a half-life of about 1 hour, and it is eliminated quickly. As such, it is usually paired with an oral compound that circumvents the issue of resistance, and prevents recurrence. What are these combinations?

• Artemether + Lumefantrine
• Artesunate + Amodiaquine
• Artesunate + Mefloquine
• Dihydroartemisin + piperaquine
• Artesunate + Sulfadoxine-Pyrimethamine

Data for their use is quite favorable. In a Cochrane review of 14 randomized trials (29), ACT was associated with improvement in parasitemia at 24hrs (RR 0.42, 95% CI 0.36-0.50) and fevers (RR 0.55, 95% CI 0.43-0.7) when compared to chloroquine. In the same review, the combination of dihydroartemisinin-piperaquine was associated with decreased recurrent parasitemia at day 28 (RR 0.20, 95% CI 0.08-0.49). Another Cochrane review of 27 studies (30) also found that DHA-P, when compared to artemether-lumefantrine, was associated with lower treatment failure at day 28 (RR 0.34, 95% CI 0.30-0.39) . This shouldn’t be surprising, as it is the active metabolite of artemisinin derivates and it has the majority of the anti-parasitic activities.

Duration of therapy was determined early on, with one randomized trial (32) finding that treatment with 3 days of artesunate provided significantly more efficacy in terms of treatment failure when compared to one day therapy and control with sulfadoxine-pyrimethamine:

The WHO analyzed four randomized controlled trials (31) and found that three days of artesunate reduced PCR-adjusted treatment failure rate within the first 28 days from that of those treated with one day (RR 0.45, 95% CI 0.36-0.55) as well as reducing the number of patients that had gametocytemia at day 7 (RR 0.74, 95% CI 0.58-0.93). This also held true for non-falciparum malaria species. WHO analysis found that ACTs cleared parasites from the peripheral blood more quickly (RR 0.42, 95% CI 0.36-0.50).

I mentioned previously that the fact that P.vivax and P.ovale tend to “hide” in the liver. ACT does not act on parasites in the liver. Because of this, the PO agent needs to be able to target this phase of the parasite that is in the liver. A Cochrane review of 14 studies (33) found that primaquine for P. vivax was associated with improvement in parasitemia with a 14 day therapy compared to 5 days:

Further, Primaquine was found to be associated with reduced relapse at 15 months of follow up if treated with 14 days of it (RR 0.60, 95% CI 0.48-0.75). One caveat with this is G6PD deficiency, for which you may need to modify the dosing depending on the activity of G6PD. One of the new agents includes tafenoquine, which is related to primaquine and is able to be dosed once a day (43), however it is still contraindicated in G6PD deficiency.

What about severe malaria?

WHO guidelines note that severe malaria involves any of the following:

WHO guidelines (31) note that artesunate therapy for at least 24hrs until patients can tolerate PO therapy, followed by 3 days of ACT is reasonable. This is based on a review (34) of 8 trials that found that artesunate was associated with reduced risk of death (RR 0.61, 95% CI .50-0.75). In other words, therapy for severe malaria is not terribly different form uncomplicated malaria, however it is different that it may take a few more days of IV therapy with artesunate depending on how many days it takes to get to PO therapy. One of the modalities touted are plasma exchange. The idea behind this is exchanging bad RBCs for normal ones. Data for this, however, is not great. A retrospective study of 817 patients (35) performed propensity score matching found mortality between groups was not significantly different (17.8% in plasma exchange vs 15.9% in no exchange) with no significant association between plasma exchange and survival (OR 0.84, 95% CI 0.44-1.60). Another meta-analysis (36) of 8 case-control studies found no benefit of exchange transfusion compared to antimalarial chemotherapy alone:

To summarize:

• ACT is a reasonable option upfront for all malaria. You can use HCQ if there is no resistance
• 3 days of arteminisin-based IV therapy with 14 days of PO therapy for uncomplicated malaria is reasonable
• IV therapy as above for severe malaria is also reasonable. Once they’re able to take PO, an additional 3 days of artemisinin followed by 14 days of PO therapy is reasonable
• There is little data to support plasma exchange.

Treatment – Babesia

Guidelines from the IDSA recommend using either atovaquone + azithromycin OR clindamycin + quinine for 7-10 days (37). An open label trial (38) found no difference in duration of babesia infection between atovaquone/azithromycin and clindamycin/quinine:

This was also corroborated in a review (39) which is based mostly on case reports and case series:

Exchange transfusion has been used in babesia, with reviews suggesting it being use with parasitemia >/= 10%, or with severe hemolysis of pulmonary/renal involvement (39). A case review (40) found an average reduction of parasitemia of 74% with exchange transfusion. Duration depends, but likely once parasitemia is down to 5% is reasonable (41), which is supported by the apharesis guidelines (42). Unfortunately, there is not much data here, so exchange transfusion may be more commonly used in severe Babesiea due to lack of good data. This may be a reasonable option in those who are severely ill and are getting therapy with antibiotics, with the same idea as exchange transfusion for malaria applying here.

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