malaria and dengue – most common mosquito-borne tropical … · 2016-11-30 · innersense 179 may...

Innersense 179 May 2016

Malaria and Dengue – Most Common Mosquito-borne Tropical Infections 1

Malaria and Dengue – Most Common Mosquito-borne Tropical Infections

Tropical diseases, according to World Health Organization (WHO) encompass all diseases that occur solely, or principally, in the tropics. The term refers to infectious diseases that thrive in hot, humid conditions, such as malaria, leishmaniasis, schistosomiasis, onchocerciasis, lymphatic filariasis, Chagas disease, African trypanosomiasis, and dengue. We would be discussing the two most common mosquito borne infections in India – malaria and dengue. Malaria (1, 2, 3, 4) Malaria is a major public health problem in India, accounting for sizeable morbidity, mortality and economic loss. India reports around 1.5 million malaria cases annually. Malaria is a parasitic disease that is passed from one human to another by the bite of infected Anopheles mosquitoes. In India, Plasmodium falciparum and P. vivax are the most common species causing malaria, their proportion being around 50% each. P. vivax is more prevalent in the plain areas, while P. falciparum predominates in forested and hilly areas. However, malaria caused by P. falciparum is serious; any delay in treatment may lead to consequences including death. Laboratory Diagnosis According to National Vector Borne Disease Control Programme (NVBDCP) and National Institute of Malaria Research (NIMR), all clinically suspected malaria cases should be investigated immediately by microscopy and/or Rapid Diagnostic Test (RDT). Microscopy Microscopy of stained thick and thin blood smears remains the gold standard for confirmation of diagnosis of malaria. It identifies different malaria-causing parasites (P. falciparum, P. vivax, P. malariae and P. ovale), their various parasite stages, including gametocytes, and the quantification of parasite density to monitor response to treatment. It is the method of choice for the investigation of malaria treatment failures. Advantages of microscopy are high sensitivity and detection of malaria parasites at low densities. In a febrile patient, three negative malaria smears 12–24 hours apart rules out the diagnosis of malaria. Rapid Diagnostic Test (RDT) RDT are based on the detection of circulating parasite antigens. Some of them can only detect P. falciparum, while others can detect other parasite species also. The NVBDCP has recently rolled out bivalent RDTs (for detecting P. falciparum and P. vivax) for use in the public health sector. Such immunologic ("immunochromatographic") tests most often use a dipstick or cassette format, and provide results in 2-15 minutes and are used in locations where microscopy results are not obtainable within 24 hours of sample collection.. It should be noted that Pf HRP-2 based kits may show positive result up to three weeks after successful treatment and parasite clearance. In these cases, results should be correlated with microscopic diagnosis. Also, it is recommended that all RDTs are followed-up with microscopy to confirm the results and if positive, to quantify the proportion of red blood cells that are infected. How Should Malaria in Pregnancy be Diagnosed? RDT should not replace blood films. RDT may miss low parasitaemia, which is more likely in pregnant women, and RDT are relatively insensitive in P. vivax malaria. A positive RDT should be followed by microscopy to quantify the number of infected red blood cells and to confirm the species and the stage of parasites.



Other Laboratory Diagnostic Methods Serology Serology detects antibodies against malaria parasites, and does not detect current infection but rather measures past exposure. Routine Tests In addition to ordering malaria specific diagnostic tests, complete blood count and a routine chemistry panel should be conducted. In case the person does have a positive malaria test, these additional tests will be useful in determining whether the patient has uncomplicated or severe manifestations of the malaria infection. Specifically, these tests can detect severe anemia, hypoglycemia, renal failure, hyperbilirubinemia, and acid-base disturbances. Dengue (5, 6, 7, 8) Dengue virus belongs to family Flaviviridae, having four serotypes; DV-1, DV-2, DV-3 and DV-4, that spread by the bite of infected Aedes mosquitoes. It causes a wide spectrum of illness from mild asymptomatic illness to severe fatal dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS). It is the most rapidly spreading mosquito-borne viral disease of mankind, with a 30-fold increase in global incidence over the last five decades. Dengue virus was isolated in India for the first time in 1945. The first evidence of occurrence of dengue fever (DF) in the country was reported in 1956 from Vellore district in Tamil Nadu. The first DHF outbreak occurred in Calcutta (West Bengal) in 1963. Of the 36 states/union territories, 35 (all except Lakshadweep) have reported dengue cases during the last two decades. In India, Ae. aegypti is the main vector in most urban areas; however, Ae. albopictus is also incriminated in many states.

States/Districts that reported dengue cases since 1991 in India Case Definition

Dengue Case Classification



Probable DF/DHF: A case compatible with clinical description of DF during outbreak

OR Non-ELISA based NS1 antigen/ IgM positive (A positive test by RDT will be considered as probable due to poor sensitivity and specificity of currently available RDTs.) Confirmed Dengue Fever: A case compatible with the clinical description of dengue fever with at least one of the following: Isolation of the dengue virus (Virus culture+VE) from serum, plasma, leucocytes. Demonstration of IgM antibody titre by ELISA positive in single serum sample. Demonstration of dengue virus antigen in serum sample by NS1-ELISA. IgG seroconversion in paired sera after 2 weeks with four-fold increase of IgG titre. Detection of viral nucleic acid by PCR. Grading of DF/DHF DF: Fever of 2-7 days with two or more of following- headache, retro orbital pain, myalgia, arthralgia with or without leukopenia, thrombocytopenia and no evidence of plasma leakage. DHFI: Above criteria plus positive tourniquet test and evidence of plasma leakage. Thrombocytopenia with platelet count less than 100000/cu mm and Hct rise >20% over baseline. DHFII: Above plus some evidence of spontaneous bleeding in skin or other organs (black tarry stool, epistaxis, gum bleeds) and abdominal pain. Thrombocytopenia with platelet count less than 100000/cu mm and Hct rise >20% over baseline. DHFIII (DSS): Above plus circulatory failure (weak rapid pulse, narrow pulse pressure <20 mm Hg, hypotension, cold clammy skin, restlessness). Thrombocytopenia with platelet count less than 100000/cu mm and Hct rise >20% over baseline. DHFIV (DSS): Profound shock with undetectable blood pressure or pulse. Thrombocytopenia with platelet count <100000/ cu mm and Hct rise >20% over baseline. Testing Algorithms for Dengue a. PCR DENV can be detected in the blood from patients for approximately the first 5 days of symptoms. Because antibodies are detected later, RT–PCR has become a primary tool to detect virus early in the course of illness. Current tests are between 80-90% sensitive, and more that 95% specific. A positive PCR result is a definite proof of current infection and it usually confirms the infecting serotype as well. However, a negative result is interpreted as "indeterminate". Patients receiving negative results before 5 days of illness are usually asked to submit a second serum sample for serological confirmation after the 5th day of illness (refer dengue diagnostic process below).

Dengue Diagnostic Process

b. MAC ELISA IgM antibody capture ELISA (MAC-ELISA) is based on capturing human IgM antibodies on a microtiter plate using anti-human-IgM antibody followed by addition of dengue virus specific antigen (DENV1-4). MAC-ELISA has a sensitivity and specificity of approximately 90% and 98%, respectively but only when used five or more days after onset of fever (i.e., in convalescent phase). One of the limitation of this testing is the cross reactivity between other circulating flaviviruses, which must be considered when working in regions where multiple flaviviruses co-circulate. IgM detection is not useful for dengue serotype determination due to cross-reactivity of the antibody.



c. IgG ELISA It is used for detection of a past dengue infection & utilizes same viral antigens as MAC ELISA. This assay correlates with the hemagglutination assay (HI) which was previously used; however, it lacks specificity within the flavivirus serocomplex groups. Primary versus secondary dengue infection can be determined using a simple algorithm. Samples with a negative IgG in the acute phase and a positive IgG in the convalescent phase of the infection are primary dengue infections. Samples with a positive IgG in the acute phase and a 4-fold rise in IgG titer in the convalescent phase (with at least a 7 day interval between the two samples) is a secondary dengue infection (refer dengue diagnostic process).

Primary DENV Infection Secondary DENV Infection

d. NS1 ELISA The non-structural protein 1 (NS1) of the dengue viral genome has been shown to be useful as a tool for the diagnosis of acute dengue infections. Dengue NS1 antigen has been detected in the serum of DENV infected patients as early as 1 day post onset of symptoms (DPO), and up to 18 DPO. NS1 assay is also useful for differential diagnostics between flaviviruses because of the specificity of the assay.

Advantages and Limitations of Dengue Diagnostic Methods

Diagnostic Tests Advantages Limitations

Nucleic acid detection

•Most sensitive and specific •Possible to identify serotype •Early appearance (pre-antibody), so opportunity to impact on patient management

•Potential false positive due to contamination •Not possible to differentiate between primary and secondary infection

Isolation in cell culture and identification using immuno-fluorescence

•Specific •Possible to identify serotype by using specific antibodies

•Takes more than 1 week •Not possible to differentiate between primary and secondary infection

Antigen detection in clinical specimens

•Easy to perform •Opportunity for early diagnosis may impact on patient treatment

•Not as sensitive as virus isolation or RNA detection

Serologic tests: IgM tests Seroconversion: 4-fold rise in HI or ELISA IgG titres between acute and convalescent samples

•Useful for confirmation of acute infection •Least expensive •Easy to perform •Can distinguish between primary and secondary infection

•May miss cases because IgM levels may be low or undetectable in some secondary infections •Need two samples •Delay in confirming diagnosis

NVBDCP-recommended Tests for Laboratory Diagnosis For confirmation of dengue infection, Government of India (GoI) recommends use of ELISA-based antigen detection test (NS1) for diagnosing the cases from the first day onwards and antibody detection test IgM capture ELISA (MAC-ELISA) for diagnosing the cases after the fifth day of onset of disease.



Conclusion Mosquito-borne infections seem to be difficult to diagnose, since it can be mimicked by other common infections that share the same endemic geography. Efficient and accurate diagnosis of malaria and dengue are of primary importance for clinical care (i.e. early detection of severe cases, case confirmation and differential diagnosis with other infectious diseases), surveillance activities, outbreak control, pathogenesis, academic research, vaccine development, and clinical trials. Optimal control of laboratory investigation process and increased awareness of possible inaccuracy in diagnosis of these two infections will help improve the diagnosis and treatment of dengue infection. References 1. Guidelines for Diagnosis and Treatment of Malaria in India; NIMR; NVBDCP 2014 2. Malaria Diagnosis; CDC 2015 3. RCOG Green–top Guideline No. 54b; 2010 4. WHO http://www.who.int/en/; 2016 5. National Guidelines for Clinical Management of Dengue Fever; WHO GoI; NVBDCP 2014 6. Dengue; Centers for Disease Control and Prevention 2016 7. Handbook for Clinical Management of Dengue; ISBN 978 92 4 150471 3; WHO TDR 2012 8. Peeling RW et al. Nature Rev Micro; Dec 2010: S30-38

Tests Offered at SRL

Test Name Method Test Code

FEVER SCREENING PANEL (CBC, ESR, CRP, BLOOD CULTURE/ SENSITIVITY, RAPID TYPHI IgM, MALARIA AG DETECTION & URINALYSIS)

CELL COUNTER/ MODIFIED WESTERGREN/ BACTEC CULTURE/

RAPID IMMUNOASSAY/ NEPHELOMETRY/ DIPSTICK &

MICROSCOPY

5012

MALARIA ANTIBODY IMMUNOCHROMATOGRAPHY 1394 MALARIA ANTIGEN (P. FALCIPARUM) DETECTION IMMUNOCHROMATOGRAPHY 1395

DENGMAL (Dengue Abs (Rapid), MP, CBC) IMMUNOCHROMATOGRAPHY/

MICROSCOPY/ AUTOMATED CELL COUNTER

DT3210

DENGUE DUO RAPID TEST IMMUNOCHROMATOGRAPHY 7599R DENGUE NS1 ANTIGEN TEST ENZYME IMMUNOASSAY 7648 DENGUE RNA PCR REVERSE TRANSCRPTASE PCR 7602 DENGUE VIRUS IgG & IgM ANTIBODIES, QUALITATIVE IMMUNOCHROMATOGRAPHY 7600

DENGUE VIRUS IgG & IgM ANTIBODIES ENZYME IMMUNOASSAY 7601, 7646

http://www.who.int/en/

malaria and dengue – most common mosquito-borne tropical … · 2016-11-30 · innersense 179 may...

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