GABGAB

23 october 200723 october 2007

Parasitology, 2007?

M. LontieMCH, Leuven

Ocular micrometer disk

• each objective must be calibrated with reference material

• can be roughly checked with a counting chamber, with RBCs ...

Eosinophilia > 10%With helminths,

insects (myasis),

not with protozoa excepting Isospora belli and Dientamoeba

fragilis (with pinworms?)

Auto-infectious

Enterobius vermicularis

Hymenolepis nana

Strongyloides stercoralis

Threats

• Global warming – El Nino (little child in Spanish, arriving around

Christmas).

• Dams – Diama dam in Senegal and large outbreak of

Schistosoma mansoni infections. 1996. Kongs et al. Trop Med Int Health. 191-8.

– The Lancet. 2001. 570-1.

IatrogenicHepatitis C in Egypt and mass treatment of schistosomiasis

• Rao M. et al. 2002. BMC Infect Dis: 29.

Nile delta of Egypt: 1950-1980.

Injections of tartar emetic (antimony potassium tartrate) with inadequately sterilized needles and syringes.

M. Wéry, 1995.

Kovats R. et al. 2003. The Lancet . El Nino and health

Kovats R. et al. 2003. The Lancet . El Nino and health

Specific gravities

• S.G. of Zn-sulfate 33 %: 1.180• S.G. of formol-solution 10 %: 1.019• S.G. of ether: 0.714• S.G. of parasites: Ancylostoma 1.055; Giardia

1.060; Entamoeba histolytica (coli) and Endolimax nana 1.065 - 1.070; Ascaris 1.110; Trichuris 1.150, Chilomastix mesnili 1.180; Ascaris (unfertilized) 1.200 (Bailenger, 1965).

Entamoeba histolytica Trophozoite (magna variety) in faeces. Diameter

approximately 30 m. Nucleus with typical fine chromatin picture (iron-hematoxylin stain).

Entamoeba histolytica Trophozoite (magna variety) in faeces. Diameter

approximately 30 m. Nucleus with typical fine chromatin picture and central karyosome (Lugol stain).

Entamoeba histolytica Trophozoite (minuta variety) in faeces. Diameter

approximately 15 m. Nucleus with typical fine chromatin picture (iron-hematoxylin stain).

trophozoiteimmature

cystmature

cyst

Entamoeba histolytica

Entamoeba histolytica Cyst in faeces with three visible nuclei. In the nucleus

at the right we clearly see the central karyosome (Lugol stain).

Entamoeba histolytica Cyst in faeces with two visible nuclei. In the nucleus at

the left we clearly see the central karyosome (Lugol stain).

Entamoeba histolytica Cyst in faeces with one visible nucleus with a central

karyosome (Lugol stain).

Entamoeba histolytica Cyst in faeces with one visible nucleus with a central

karyosome (Lugol stain).

Entamoeba histolytica Cyst in faeces with one visible nucleus with a central

karyosome (Lugol stain).

Entamoeba histolytica Cyst in faeces with one visible nucleus with a central

karyosome (Lugol stain).

Entamoeba histolytica Cyst with one visible nucleus and one cylindrical

chromatoidal body (Lugol stain).

Entamoeba histolytica Cyst with two nuclei and one cylindrical chromatoidal

body (Lugol stain).

Entamoeba histolytica Cyst with one visible nucleus and one cylindrical

chromatoidal body (Lugol stain).

Entamoeba histolytica - dispar

• Entamoeba polecki Entamoeba hartmanni (small race E. histolytica)

• Entamoeba histolytica Laredo strain Entamoeba dispar non pathogenic strains grow betweeen 20 and

37°C, pathogenic only at 37°C isoenzyme analysis: only 9 zymodemes are

pathogenic

Entamoeba histolytica - dispar• PCR, isoenzyme analysis, and antigen detection

(JCM, 1998, 449).

• Monoclonal antibodies (JCM, 2001, 716).

• ITG-Antwerp: PCR under evaluation (T. Vervoort): fecal material in 3.5% formalin.

Entamoeba coli Large cyst (> 20m) in faeces with four visible nuclei

(Lugol stain).

Dientamoeba fragilis

• Two nuclei.• “The unflagellated human

flagellate “.• Only (very labile)

trophozoites, no cysts.• Questionable enteric

pathogen.• Doxycycline,

paromomycin, metronidazole (Sanford et al., 2005).

Courtesy CDC

Dientamoeba fragilis

SAF fixative (sodium acetate acetic acid formalin) and iron hematoxylin stain have replaced the PVA (polyvinyl alcohol fixative with HgCl2) and trichrome stain.

Sodium acetate acetic acid formalin (SAF)

• Sodium acetate 1.5 g

• Acetic acid, glacial 2.0 ml

• Formaldehyde, 37-40 % 4.0 ml

• Distilled water 92.0

ml

Dientamoeba fragilisIn saline (unstained).

Dientamoeba fragilisIn saline (unstained).

Dientamoeba fragilisIn saline (unstained).

Dientamoeba fragilis

• In The Netherlands.• In 247 unpreserved

stool specimens: none.• In 247 SAF-preserved

stool specimens: 24.

(Mank T. 1997. Thesis)

Stained with hematoxylin.

Dientamoeba fragilis

• In Brussels, Belgium.• SAF-preserved stool

specimens used.• D. fragilis (6.3%) and

G. lamblia (7.1%) in 448 patients.

Vandenberg O. et al. 2006. Int J Infect Dis:221, 2.

Stained with hematoxylin.

Dientamoeba fragilis

MCH, Leuven• (2005-2007)• 6 + / 160 SAF

Stained with hematoxylin.

Blastocystis hominis Cyst in faeces (Hematoxylin stain).

AntoonvanLeeuwenhoek

Giardia lamblia1681

Giardia lamblia Trophozoite with two nuclei and several flagella

(May-Grünwald-Giemsa stain)

Giardia lamblia: antigen detection by IF and ELISA

• monoclonal antibodies: Merifluor (MERIDIAN) (Cryptosporidium and Giardia).

• 8/9 Giardia ELISAs are OK. (JCM, 1998, 1338).

• Triage parasite panel (BIOSITE) useful. (JCM, 2000, 3337; JCM, 2001, 334).

• one ELISA almost as sensitive as two microscopic examinations. (Mank T. 1997).

Antigen detection

• Good sensitivity and specificity

• In combination with conventional tests

• Problem = COST (Triage = $ 19.44/test)

Leishmaniasis

• Leishmania infantum, Leishmania donovani, … in macrophages of man.

• Female sandflies: Phlebotomus spp., Lutzomia spp.,...

• Cutanous, visceral leishmaniasis (Kala-azar).• Fever, hepatosplenomegaly, pancytopenia.• Serology - parasitological examination of

bone marrow aspiration.

(A. Van Gompel et al., T v G, 1997)

Leishmania sp. Ovoid small (2-6 m) intracellular parasite in a bone marrow

aspirate. The typical rod shaped kinetoplast is seen besides the nucleus (May-Grünwald-Giemsa stain).

Baghdad-boil, 2004

• Walter Reed Army Medical Center, Washington.

• Several hundred US soldiers in Iraq.

• Mostly cutaneous, but also visceral forms.

Diagnosis of malaria

• Geographical distribution of different species should be taken into account.

• Plasmodium falciparum limited to (sub-) tropical areas (summer isotherm of 20°C, altitude < 2000 m).

• Mixed infections are not uncommon (eg P. falciparum + P. ovale in West Africa).

Duffy blood group system = receptor P. vivax

West & Central Africa Duffy bg: absent Plasmodium ovale

East Africa Duffy bg: present Plasmodium vivax

Wéry M. 1995. Protozoologie Médicale.

vivaxovale falciparum

falciparummalariae

vivax

falciparumvivaxmalariae

(vivax)(malariae)

(falciparum) (vivax)

MALARIA

ovale

malariae

According to M. Wéry, 1995

Parasite antigen detection

Lynne S. Garcia *

At the ASM-meeting, Salt Lake City, May 2002.

– Absence of trained personnel.

– Usefull screening assays.

* Author of Practical Guide to Diagnostic Parasitology, ASM Press.

Malaria: antigens

• HRP-2: watersoluble histidine-rich protein from axesual stages and young gametocytes of P. falciparum (related to knob-associated HRP-1 and HRP-3).

• Pan-malarial antigen: pLDH or aldolase, present in all four Plasmodium spp.

A. Moody. 2002. CMR, 15:66-78.

• Optimal-IT: Flow Diagnostics and Dia Med

– HRP2– pLDH

• Binax-ICT: Herman Diagnostics

– HRP2– aldolase ( Parasight-F: not in Belgium)

Malaria antigen (HRP2) detection

Sensitivity: 50-100 (and less) parasites / µL.

(No false negatives due to very high parasitemias.)

Diagnosis of malaria antigen-detection

• Persistent positivity with gametocytes (JCM, 2001, 1025).

• PMA is expressed by P. malariae (JCM, 2001, 2035).

• Moody A. 2002. CMR, 15:66-78. Rapid diagnostic tests for malaria parasites (review).

ICT, Binax & Optimal, Playford E. & Walker J. 2002. JCM, 40:4166-4171.

ICT Binaxo P. f: 32+ / 33o P. v.: 22+ / 50o P. v. (1000-10 000)

False Negative: 12 /25o Overall specificity:

96.8%

Optimalo P. f: 28+ / 33o P. v.: 40+ / 50o P. v. (1000-10 000)

False Negative: 4 / 25o Overall specificity:

98.4%

ICT, Binax, USA

• Richter J. et al. 2004. Parasitol Res. 94:384-385. – Monoparasitic and mixed P. falciparum

infections detected : 56/56 – Monoparasitic infections detected:

P. vivax: 3/8

P. ovale: 0/3

P. malariae: 0/2

ICT, Binax, ineffective for P. ovale

• Bigaillon C. et al. 2005. JCM.– French military hospital, 2002-2004– Reference = PCR ( microscopy)

• P. vivax: 9 / 9

• P. ovale: 3 / 12

• P. malariae: 1 / 1

• P. falciparum: 92 / 93

Lee N. et al. 2006. JCM., 44:2773-2778.

Effect of sequence variation in Plasmodium falciparum histidine-rich protein 2 on binding of specific monoclonal antibodies: implications for rapid diagnostic tests for malaria.

• Sequence variation can help to explain the variations in the performance of HRP-based RDTs and point toward possible solutions.

Limited level of accuracy provided by available rapid diagnosis tests for

malaria enhances the need for PCR-based reference laboratories

Rubio JM et al. 2001. JCM, 2736-7.• 169 patients in Spain.

• ParaSight-F, OptiMal, ICT Pf/Pv.

• Microscopy and semi-nested PCR.

Species-specific PCR diagnosis of malaria (CDC)

1. P. vivax 2. P. malariae 3. P. falciparum 4. P. ovale

Two step nested PCR using the primers of Snounou et al. CDC, 2001.

Detection of four Plasmodium species in blood from humans by

18S rRNA gene subunit-based and species-specific real time PCR

assays

Rougemont M. et al. 2004. JCM, 42:5636-5643.

Detection of four Plasmodium species in blood from humans by

18S rRNA gene subunit-based and species-specific real time PCR

assays

Rougemont M. et al. 2004. JCM, 42:5636-5643.

Malarone

• Atovaquone + proguanil.

• For prophylaxis and treatment.

• Probable effect on hypnozoïtes.

• Experience increasing.

• (Expensive).

Malarone

A systematic review and meta-analysis …

• Evidence shows that Malarone is highly efficacious as a prophylactic agent and is very well tolerated.

Nakato H. et al. 2007. JAC:929-936.

Artemisia annua

• Chinese herb.

• Artemether, artenusate and artemisin derivatives (Qinghaosu).

• Short half-life.

• Fast acting.

• In China in combination with lumefantrine (1970s in Beijing).

Artenusate versus quinine for treatment of severe falciparum

malaria: a randomised trial.

South East Asian Quinine Artenusate Malaria Trial group.

Correspondence: Prof. N.J. White, Thailand.

The Lancet. 2005. 366:717:725.

Interpretation: artenusate should become the treatment of choice for severe falciparum malaria in adults.

WHO. 2001. Antimalarial drug combination therapy

• Amodiaquine + sulfadoxine-pyrimethamine

• Amodiaquine + artenusate

• Artemeter-lumefantrine (Co-Artem)

Baird J.K. 2005. NEJM 352:1565-1577Combined therapies

• Treatment of leprosy, tuberculosis, HIV, ...

• Chloroquine and sulfadoxine-pyrimethamine

• Amodiaquine and artenusate

• Lumefantrine and artemether

• Artemisin and mefloquine

Artemisin combination treatment (ACT)

• Drugs for Neglected Diseases Initiative and Sanofi-Aventis.

• Fixed dose combination of artenusate and amodiaquine (2006).

• Two tablets per day for three days.

• Target price per treatment course: US $1 ($0.50 per child).

Pam Das. p. 267 in The Lancet Infectious Diseases of May 2005.

Mutabingwa T. et al. 2005. The Lancet 365:1474.

Baird J.K. 2005. NEJM 352:1565-1577Parasite Burden

• Patients with malaria: 108 to 1013 parasites.

• Artemisin: reduction of 104 with each cycle.

• Three days artemisinin: reduction of 108.

• Tetracycline: reduction of 101 with each cycle.

Baird K. 2OO5. NEJM. 352:1565.Artemisin + mefloquine

Resistance of Plasmodium falciparum field isolates to in-vitro artemether and

point mutations of the SERCA-type PfATPase.

Jambou R. et al. The Lancet. 2005. 366:1960-1963.• Cambodia, French Guiana, Senegal.• All resistant isolates came from areas with

uncontrolled use of artemisinin derivatives.• Need for rapid deployment of drug combinations.

Prevalence of in vitro resistance to eleven standard or new antimalarial drugs among

Plasmodium falciparum isolates from Point-Noire, Republic of Congo

• Pradines B. et al. 2006. AAC, 44:2404-2408.• 40-110 isolates tested with isotopic microtests.• No resistance to halofantrine, lumefantrine,

dihydroartemisinin, atovaquone, • Resistance to chloroquine (75.5%), quinine (6%),

monodesethylamodiaquine (2%), mefloquine (7%), cycloguanil (36%), pyrimethamine (68%).

Arrow K. et al. 2005. NEJM. Mpos58168.

• The need for general use of artemisinin-based combination therapies is by now universally accepted.

• Goal: price for an adult < $1.

• Must be compatible with the current market-driven distribution system.

WHO requests halt of sales of monotherapy drugs for malaria

Roxanne Nelson. 2006. p 132. In The Lancet Infectious Diseases, Volume 6,

Issue 3.

Eradication of malaria

1934: Hans Andersag at Bayer discovers chloroquine1939: Paul Müller at Geigy discovers DDT1951: Sardinia malaria free1955: WHA (WHAssembly): goal of global eradication1955-1969: WHO uses DDT and chloroquine50’s: DDT-resistance1962-1970: chloroquine-R1955-1965: expenditure of $ 1.4 billion1969: WHO back to malaria control1975: Europe free of malaria for first time in history

Courtesy of C.D.C