The Diagnosis of Malaria Norman Moore, PhD Director of Medical Affairs

The majority of the statistics and facts used in this presentation are available on the Centers for Disease Control Website.

Historical Perspective Malaria has been around for a minimum of 4,000 years China described the symptoms in 2700 BC Thought responsible for the decline of many Greek city-states in 400 BC The name comes from Italian for “bad air” Quinine – Comes from the bark of the Cichona tree in South America http://www.cdc.gov/malaria/about/history/ FYI – “bad air” probably because associated with swamps and stagnant water is what mosquitoes need to breed. Named after the countess of Cichona who was treated by indigenous Indian tribes in Peru

Malaria Parasite Parasite first discovered in 1880 by a French army surgeon by seeing the parasites in blood. A British officer in the Indian Medical Reserve in 1897 showed that the parasite could be passed to mosquito from person. He later showed it could go bird-to-bird through mosquito vector. Life cycle shown in 1898-1899 when mosquitoes that had bitten a malaria patient in Rome were sent to London and able to infect volunteers there. www.cdc.gov/malaria/about/hstory/

Anopheles Mosquito the Anopheles sp. vector ingests the gametocytes, microgametocytes develop into microgametes, which are able to fertilize gametes.

US Malaria Issues Panama Canal – Out of the 26,000 people working on the canal, 21,000 were hospitalized with malaria at some point. Construction only possible after the control of malaria and yellow fever put in. US Public Health Service started controlling malaria in US around 1914. Tennessee Valley Authority had authority to control malaria. When it started in 1933, 30% of the population was affected and by 1947, it was eradicated. CDC and states started eradication program in 1947 and it was considered eliminated in 1951. www.cdc.gov/malaria/about/history

Malaria in the US Approximately 1,500 cases of malaria are reported each year in the US. Cases are due to travel to endemic regions and immigration. Since 1957, there have been 63 reported outbreaks of malaria in the US. Occurred by local mosquitoes biting an infected person. The mosquito species previously responsible for disease in the US are still here. Since 1963, there have been 96 cases of transfusion-transmitted malaria. Donors SHOULD defer depending on where/when they have been in malaria-infected regions. www.cdc.gov/malaria/about/facts.html

Malaria in the World 3.3 billion people live in malaria regions Half of the world’s population In 2008, estimated that there were 190-311 million cases with 708,000- 1,003,000 deaths. 89% of the deaths occurred in Africa. Malaria is the 5th most common death due to infectious disease worldwide. www.cdc.gov/malaria/about/facts.html Top four deaths due to infectious disease are respiratory, HIV/AIDS, diarrheal disease, and tuberculosis.

Life Cycle Two types of hosts – humans and female Anopheles mosquitoes. In humans, the parasite first grows in liver cells and then in red blood cells. In the blood cells, parasites grow, lyse the cell, and then other red blood cells infected. In mosquitoes, if gametocyte ingested, a new cycle is initiated. 10-18 days later, sporozoite is found in mosquito’s salivary glands. Can be injected in person and go to liver cells.

Life Cycle Note: P. vivax and P. ovale can lie dormant in www.cdc.gov/malaria/about/biology Note: P. vivax and P. ovale can lie dormant in liver for weeks or even years.

Types of Malaria Affecting Humans Plasmodium falciparum – most virulent P. vivax P. ovale P. malariae Rare cases of P. knowlesi

Disease Symptoms vary dramatically None to very mild to severe/death Toxins are dumped into the bloodstream when the infected red blood cells lyse. Some toxic factors stimulate the macrophages to produce cytokines that in turn produce fever and shaking. Red blood cells infected with P. falciparum don’t freely circulate. When they sequester in the brain, can cause cerebral malaria, which has high mortality. www.cdc.gov/malaria/about/disease.html

Incubation Period After acquiring Plasmodium, the incubation period ranges from 7 to 30 days. Shorter incubation periods usually associated with P. falciparum, while longer ones can be P. malariae. Prophylactic anti-malarial drugs may delay symptoms for weeks to months. May result in missed or delayed diagnosis. Patients should let health care professionals know if they have traveled to malaria endemic regions during the last 12 months. www.cdc.gov/malaria/about/disease.html Dormant is more likely with P. vivax and P. ovale.

Uncomplicated Malaria Symptoms “Classic” attack (rarely observed – lasts 6-10 hours) Cold stage (patient feels cold, shivering) Hot stage (fever, headache, vomiting, seizures in young children) Sweating stage (sweat, then return to normal temp) Common symptoms Fever, chills, sweats, headache, nausea & vomiting, body aches, and general malaise If in non endemic region, these symptoms could be confused for other sicknesses If in endemic region, may believe it is malaria without checking other causes

Severe Malaria Symptoms Cerebral malaria – unconscious, seizure, coma, other neurological difficulties Severe anemia Blood in the urine Acute respiratory distress syndrome (ARDS) Problems with blood coagulation Kidney failure Hypoglycemia www.cdc.gov/malaria/about/disease.html ARDS has inflammation reaction in the lungs that inhibits oxygen exchange, which may occur even after the parasite counts have decreased in response to treatment

Diagnosis of Malaria

Specimen Collection Ideally, blood can be collected by finger prick If other tests being performed, can use venipuncture EDTA is preferred as the anticoagulant as heparin may lead to morphological distortion Smears should be prepared and stained within an hour of drawing the specimen. Alterations in morphology may occur if delayed. Manual of Clinical Microbiology “Diagnosis” pg. 2041

Information to Collect Travel history (Time between bite & symptoms) Help suggest likelihood of infection 9 to 14 days for P. falciparum, P. vivax, and P. ovale (or months for vivax and ovale) Up to 40 days for P. malariae After one month, probably not P. falciparum History of prophylaxis or treatment of malaria History of transfusion or shared needles History of malaria in person (relapse?) Knowledge of fever pattern

Microscopy – The Gold Standard Benchmark diagnostic standard for over 100 years. In expert hands: highly sensitive, specific. Results provide a wealth of clinically important data. Stained slide serves as permanent record.

Microscopy Thick film considered “gold standard” for detection of parasites due to being able to use larger volume (10µl of blood) Thin film considered “gold standard” in species identification Smear examinations should be under oil immersion Negatives should not be reported until 200 oil immersion fields have been examined Additional specimens should be examined at 12-hour intervals for a subsequent 36 hours.

Microscopy Limitations Microscopy skills may be lacking in areas not routinely doing malaria evaluations Smear preparation, staining Interpretation Mixed infections - can be difficult to diagnose. Low parasitemia - can be difficult to diagnose. Hands on time is very high.

BinaxNOW® Malaria Test

BinaxNOW® Malaria Test

Clinical Trial by US Army

BinaxNOW® Malaria Test Detects circulating malaria antigens in whole blood. 15 minute test The only FDA cleared rapid malaria test. * For parasitemia levels >5,000 (parasites/µl). Refer to product insert for additional information.

Performance Plasmodium falciparum: Plasmodium vivax: Sensitivity 95.3% Specificity 94.2% Plasmodium vivax: Sensitivity 68.9% Specificity 99.8% Note: Publications report P. ovale as being harder to detect as it only attacks young erythrocytes

How the Test Works Histidine-rich protein II (HRP II) is specific to P. falciparum while aldolase is a pan-malarial agent. Test has monoclonal antibodies directed against these proteins, which detects them in a modified lateral flow format.

BinaxNOW® Malaria Test Procedure Apply 15 µl of whole blood to the purple pad. Venous or capillary whole blood. EDTA collection tubes. Mylar-coated capillary tubes included in kit. Apply 2 drops of Reagent to the white pad below where the blood is applied. Apply 4 drops of Reagent to the pad located at the top of the left side of the test device. Close the device and read in 15 minutes.

BinaxNOW® Malaria Test TEST RESULTS DESCRIPTION / INTERPRETATION T1 Positive Positive result for P. falciparum (P.f.) T2 Positive Positive result for P. vivax (P.v.) or P. malariae (P.m.) or P. ovale (P.o.) In some cases the appearance of only the T2 Line may indicate a mixed infection with two or more of P.v., P.m., and P.o. T1 + T2 Positive Positive result for P. falciparum (P.f.) In some cases the appearance of both the T1 and T2 Lines may indicate a mixed infection of P.f. with another species. No T1 or T2 Lines Negative result (no malaria antigens were detected)

Microscopy vs. BinaxNOW® Microscopy vs. BinaxNOW Malaria TOTAL TIME: 3-6 HOURS Obtain blood Prepare thick smears Prepare thin smears Stain blood smears Microscopic examination of thick smears Microscopic examination of thin smears TOTAL TIME: 20 MINUTES Obtain blood Add blood and reagent to test device Close test device and read results in 15 minutes

Conclusions Malaria continues to be a major worldwide disease. Although it has been eradicated in the US, it is important to be able to diagnose the disease to treat as fast as possible. Microscopy remains the diagnostic gold standard, but a rapid test is FDA-cleared to help with suspected malaria cases.