**7.5. Laboratory procedures**

Blood films and rapid tests were performed from the same finger-prick blood. Blood films were dried, thin films fixed in methanol, and both films stained with 3% Giemsa for 45 minutes. Smears were read by experienced technicians, counting parasites against 200 or 500 white blood cells (WBC) or 200 high power fields before declaring a blood slide negative. The parasite density per micro-liter was calculated by multiplying the asexual parasite count by 8000 and dividing by the number of WBC counted [17].Plasmodium species were confirmed on the thin film and slides with mixed infections had only P.falciparum monoinfection, had the asexual density per microliter calculated as for P.falciparum. Gametophytes were recorded with species identification where possible. All inclusion slides were blinded and double read, with a third reading performed in case of discordance, ie; positive/negative discordance for asexual stages; asexualdensity discordance (difference in parasitemia≥50%);positive/negative gametocyte discordance. Twenty percent of the followup visit slides were also blinded and double read. External quality control of 290 inclusion slides was performed by Shokia Malaria Research Unit, Thailand, giving Mbarara laboratory, a sensitivity of 95.5% and a specificity of 100%. All RDTs were performed and interpreted according to the manufacture's instructions. Each test result was interpreted by two independent health care providers blind to the result of the blood film and reading according to a rota to avoid observer bias. The first reading was performed at the time specified by the manufacturer (15 min after preparation for Paracheck Pf and Parabank and 20 mins for Carestart and Vistapan. The second reading was performed within 15 min of the first one. Discordant results were read by the laboratory supervisor for a definitive result. Each reader also classified the test as either invalid or doubtful. A doubtful test was defined as a test for which the reader was not sure if there was any indication of a line present. At the end of the study, two test readers and two laboratory technicians involved in preparing the tests completed a questionnaire concerning the ease of use and interpretation of each test.

Analysis. All data were either recorded directly or transcribed from source data forms to an individually numbered case report form (CRF). Data were double entered and validated using EpiData version 3.1(EpiData Association,Odense, Denmark)and analysed using Stata 9.1 (Stata Corp. college station,TX,USA).The study profile and base-line characteristics were summarized, including comparative tests between age groups (x 2 test ,Ma-Whitey U test). The validity for each test was calculated overall and then stratified by age group, level of parasitemia(parasites/µl 1---99,≥100,≥200,≥500), presence/absence of fever, duration of illness(0—2vs.3 days and above) and a history of taking antimalarials, using comparative tests( x 2 test, Mann-Whitney U test) to compare differences between groups. Kappa statistics were calculated for iter-reader reliability for each test on the day of diagnosis. A test was considered as reliable if k ≥ 0.8. Univariable and multivariate analyses were performed to investigate the association between explanatory factors and the test remaining positive at each follow- up visit.

Results. Demographical and Parasitological characteristics of study subjects.

Between 26 April and 27th July, 2005, 485 patients from the out-patient department were screened. Nine were ineligible (three had severe illness, five were non-residents and one was not in the appropriate age group after completion of recruitment in the under fives. Sixteen patients did not consent to participate in the study; 239 under fives and 221 aged 5 years and above. The mean age was 12 years(SD 13years;Table 2). There were 248 positive blood films with P. falciparum monoinfections(93.6%), P. malariae monoinfections(2.4%), P. falciparum+ P. malariae mixed infections(0.8%) and P. falciparum+ P. vivax mixed infections(0.8%). Of the 212 negative films, nine had gametocytes present. Parasitological characteristics of positive subjects are given in Table 2. Slides positive with P. falciparum had higher parasite densities than those of the other two species.

Validity of RDTs.

172 Dehydrogenases

film –negative patients.

**7.4. Study procedures** 

the blood film and all RDTs

**7.5. Laboratory procedures** 

96) was doubled to permit a stratified analysis by age group (0--4 and ≥5 years. The same parameters were used to calculate the required number of patients with a negative blood film, thus giving a final minimum sample size of 200 blood-film-positive and 200 blood –

On the day of inclusion, demographic and clinical information were recorded, and a thick/thin blood film and the four rapid tests (Vistapan, Carestart, Parabank , and Paracheck-Pf) were performed. Women with positive pregnancy test and hyperparasitemic patients (P. falciparum›250,000 parasites/µl) were given quinine and excluded from further follow up. All other patients with positive blood film received an artemether-lumefantrine( Coartem®, Norvatis Pharma AG,Bassel,Switzerland), six –dose regimen under directly observed therapy. This treatment modality have been shown to be very efficacious with a prompt reversion to a negative [16] blood film after treatment. Patients receiving Coartem were asked to return to the Clinic on the third, seventh and 14th day after inclusion to repeat

Blood films and rapid tests were performed from the same finger-prick blood. Blood films were dried, thin films fixed in methanol, and both films stained with 3% Giemsa for 45 minutes. Smears were read by experienced technicians, counting parasites against 200 or 500 white blood cells (WBC) or 200 high power fields before declaring a blood slide negative. The parasite density per micro-liter was calculated by multiplying the asexual parasite count by 8000 and dividing by the number of WBC counted [17].Plasmodium species were confirmed on the thin film and slides with mixed infections had only P.falciparum monoinfection, had the asexual density per microliter calculated as for P.falciparum. Gametophytes were recorded with species identification where possible. All inclusion slides were blinded and double read, with a third reading performed in case of discordance, ie; positive/negative discordance for asexual stages; asexualdensity discordance (difference in parasitemia≥50%);positive/negative gametocyte discordance. Twenty percent of the followup visit slides were also blinded and double read. External quality control of 290 inclusion slides was performed by Shokia Malaria Research Unit, Thailand, giving Mbarara laboratory, a sensitivity of 95.5% and a specificity of 100%. All RDTs were performed and interpreted according to the manufacture's instructions. Each test result was interpreted by two independent health care providers blind to the result of the blood film and reading according to a rota to avoid observer bias. The first reading was performed at the time specified by the manufacturer (15 min after preparation for Paracheck Pf and Parabank and 20 mins for Carestart and Vistapan. The second reading was performed within 15 min of the first one. Discordant results were read by the laboratory supervisor for a definitive result. Each reader also classified the test as either invalid or doubtful. A doubtful test was defined as a test for which the reader was not sure if there was any indication of a line present. At

Only Carestart had estimates for all validity parameters greater than 90% (Table 3). Vistapan and Carestart were as sensitive as Paracheck-Pf(P=0.14 and P=0.38 respectively. Parabank was less sensitive than all other tests (P‹0.001 for each comparison). There was no significant difference in specificity between the three pLDH tests, but Parabank had a higher specificity compared with Paracheck.Pf (P= 0.02) for P. falciparum detection. Sensitivity decreased with older age for both Vistapan [97.7% (under fives) vs 85.7%, P‹0.01] and Parabank[95.4%( under fives)vs73.1%, P‹0.001]. Sensitivity increased with axillary temperature≥ 37.5 0C at inclusion for Paracheck.Pf (98.8 vs91.4%, P=0.04), Vistapan(97.6 vs 89.0%, P=0.03) and Parabank(91.8vs81.0%, P=0.04) compared with patients with axillary temperature ‹37.5 0C. Although, the small number of non-falciparum monoinfections does not permit reliable calculations of validity of non-falciparum mutants, all tests detected 100 % (n=6) of the P. malariae monoinfections. Plasmodium vivax was detected in 4/6 infections by Carestart, 2/6 by Vistapan and 1/6 by Parabank.


Functions of Dehydrogenases in Health and Disease 175

Paracheck =Pf%(95%cl)

94(233/248) [90.2-96.6]

87.3(185/212) [82.0-91.4]

89.6(233/260) [85.3-93]

92.5(185/200) [87.9-95.7]

69.7(152/218) [63.1-75.7]

8.9(19/213) [5.1-12.7]

9.5(21/221) [5.6-13.4]

4.6(9/196) [1.7-7.5]

Parabank% [95% cl)

84.7(210/248) [79.6-88.9]

94.3(200/212) [90.3-97.0]

94.6(210/222) [90.7-97.2]

84.0(200/238) [78.7-88.4]

80.8(181/224) [75.6-86.0]

3.4(51/218)[17.8- 29.0]

> 27.6( 63/228) [21.8-33.4]

8.9(18/202) [5.0-12.8]

Parameters Carestart %

Sensitivity 95.6(237/248)

Specificity 91.5(194/212)

PPV 92.9(237/255)

NPV 94.6(194/205)

Paracheck -

Carestart and Vistapan.

[95 Cl]

[90.2-96.6]

[86.9-94.9]

[89.1-95.8]

[90.6-97.3]

PPV: Positive Predictive Value; NPV: Negative Predictive Value

Pf 226 86.2(193/224)

Vistapan 221 36.1(79/219)[29.7-

Carestart 230 42.5(97/228)

Parabank 204 17.8(36/202)

Hospital, Outpatient department, SouthWestern Uganda.

Vistapan% [95%Cl)

91.9(228/248) [87.8-95]

89.6(190/212) [84.7-93.4]

91.2(228/250) [87-94.4]

90.5(190/210) [85.7-94.1]

**Table 3.** Validity of four rapid diagnostic tests for the detection P. falciiparum species in patients

[81.7-90.7]

42.5]

[36.1-48.9]

[12.5-23.1]

**Table 4.** Percentage of positive tests on each follow up visit in patients attending Mbarara Regional

refrigeration and all tests have undergone temperature stability studies up to 30 0C.

In overall, there are not large differences between the tests in terms of ease of use. Some tests have small advantages or disadvantages over others. For example, Vistapan has individual buffer sachets, considered to be an advantage, where as Carestart has a delay time of over 60 seconds between blood application and buffer application, considered to be a disadvantage. All tests results are stable for a minimum of 24hr.The number of invalid tests was ‹0.5% for Parabank and between 0.5 and 2% for Carestart and Vistapan. No test had items requiring

This study appears to be a pioneer study [17] to evaluate a new generation of pLDH tests for malaria diagnosis, performed in a mesoendemic African setting with a predominance of P. falciparum infections. The authors showed that several of these tests were validated and should be of great use in malaria endemic countries, where microscopy is not available and well trained microscopists are lacking. For confident diagnosis of malaria in routine outpatient departments, a sensitivity of more than 90% is crucial and this was achieved for

a <sup>n</sup> is the number of positive tests for each RDT on day 0 in patients who were followed up.

RDT Day 0 na Day 3% [95% Cl] Day 7 %[ 95Cl] Day 14 % [95% Cl]

attending Mbarara Regional Hospital, out-patient department, southwestern Uganda

**Table 2.** Baseline characteristics of all study subjects and Parasitological characteristics of Slide-positive subjects attending Mbarara Regional Referal Hospital, Outpatient department, South -Western Uganda

### Reliability

The k statistic for the inter-reader reliability for all tests was above 0.90(very good agreement) [Carestart,k=0.96(95.0%, Cl 0.94—0.99); Vistapan, k=0.94( 95% Cl 0.91- t 0.97); Parabank, k=0.96(95% Cl 0.94-0.99); Paracheck.Pf, k=0.97(95% Cl 0.95-1.0)]

Time to Negativity of RDTs.

There were no positive blood films on follow up visits, and therefore, every positive RDT result on day 3,7 or 14 was considered a false positive result (Table 4). All three RDTs tested had significantly fewer false positive results on every day of follow-up compared with Paracheck.Pf(P‹0.001 for all tests on day 3,7 and 14). There was o difference between the pLDH tests by day 14, with the percentage of positive tests ranging from 4.6 to 9.5%.

Younger age group and higher parasite level at inclusion were related to positive Paracheck. Pf on all follow-up days (logistic regression, P‹0.01), for all. Age group, fever at diagnosis and presence of gametocytes on day 3 were all related to a positive pLDH test on day 3 (except age group for Parabank)(age group:Vistapan P=0.026, Carestart P‹0.001.



PPV: Positive Predictive Value; NPV: Negative Predictive Value

174 Dehydrogenases

**Parameters Group A**

Gender ratio (M:F) 0.98

Mean age(SD) 2yrs

Median duration of

Fever on presentation

Asexual parasitemia

Geometric mean of

parasitemia(95%Cl)

Interquartile range (Interquartile value)

Time to Negativity of RDTs.

Gametocyte

Reliability

asexual

Previously taken

Parasitological

**(<5yrs)** 

(118:121)

(14 months)

7433(4869-

1682- 45748(44066)

Parabank, k=0.96(95% Cl 0.94-0.99); Paracheck.Pf, k=0.97(95% Cl 0.95-1.0)]

Baseline characteristics n=239 n=221 n=460

**Group B**

0.52 (76:145)

22yrs (12yrs)

illness in days (range) 3(1-14) 3(1-30) 3(1-30) 0.2(Kruskai-

antimalarial (n,%) 81(33.9) 60(27.3) 141(30.7) 0.13( X 2)

(axillary temp≥ 37.5 0C) 99(41.4) 31(14.0) 130(28.3) <0.001(X 2)

characteristic n=129 n=119 n=248 -----------

range (parasites/µl) 16-703411 16-233241 16-703411 0.001(Kruskal-

carriage(n,%) 36(27.9) 22(18.5) 58(23.4) 0.11(X 2)

**Table 2.** Baseline characteristics of all study subjects and Parasitological characteristics of Slide-positive subjects attending Mbarara Regional Referal Hospital, Outpatient department, South -Western Uganda

The k statistic for the inter-reader reliability for all tests was above 0.90(very good agreement) [Carestart,k=0.96(95.0%, Cl 0.94—0.99); Vistapan, k=0.94( 95% Cl 0.91- t 0.97);

There were no positive blood films on follow up visits, and therefore, every positive RDT result on day 3,7 or 14 was considered a false positive result (Table 4). All three RDTs tested had significantly fewer false positive results on every day of follow-up compared with Paracheck.Pf(P‹0.001 for all tests on day 3,7 and 14). There was o difference between the

Younger age group and higher parasite level at inclusion were related to positive Paracheck. Pf on all follow-up days (logistic regression, P‹0.01), for all. Age group, fever at diagnosis and presence of gametocytes on day 3 were all related to a positive pLDH test on day 3

pLDH tests by day 14, with the percentage of positive tests ranging from 4.6 to 9.5%.

(except age group for Parabank)(age group:Vistapan P=0.026, Carestart P‹0.001.

166- 11070(10904)

**(≥5yrs) Overall p-value** 

0.72

12yrs

11346) 1524(975-2384) 3475(2521-4790) 0.001(t test)

641-

23827(23186) ----------

(194:266) 0.001(X 2)

(13 yrs) N/A

Wallis)

Wallis)

**Table 3.** Validity of four rapid diagnostic tests for the detection P. falciiparum species in patients attending Mbarara Regional Hospital, out-patient department, southwestern Uganda


a <sup>n</sup> is the number of positive tests for each RDT on day 0 in patients who were followed up.

**Table 4.** Percentage of positive tests on each follow up visit in patients attending Mbarara Regional Hospital, Outpatient department, SouthWestern Uganda.

In overall, there are not large differences between the tests in terms of ease of use. Some tests have small advantages or disadvantages over others. For example, Vistapan has individual buffer sachets, considered to be an advantage, where as Carestart has a delay time of over 60 seconds between blood application and buffer application, considered to be a disadvantage. All tests results are stable for a minimum of 24hr.The number of invalid tests was ‹0.5% for Parabank and between 0.5 and 2% for Carestart and Vistapan. No test had items requiring refrigeration and all tests have undergone temperature stability studies up to 30 0C.

This study appears to be a pioneer study [17] to evaluate a new generation of pLDH tests for malaria diagnosis, performed in a mesoendemic African setting with a predominance of P. falciparum infections. The authors showed that several of these tests were validated and should be of great use in malaria endemic countries, where microscopy is not available and well trained microscopists are lacking. For confident diagnosis of malaria in routine outpatient departments, a sensitivity of more than 90% is crucial and this was achieved for Carestart and Vistapan.

The pLDH tests also demonstrated desirable qualities that could reduce the possibility of patients without malaria being treated or given antimalarial drugs, thereby reducing drug pressure and resistance, a major concern at a time when artemisinin combination therapy (ACTs) are being introduced throughout Africa. Their high specificity would reduce the number of patients with false positive results. Secondly, the great reduction in the number of tests remaining positive for a long time after treatment had been effected. This is reminiscent of the test carried out by HRP 2. Thirdly, the ability to detect both P. falciparum and P. vivax, would increase confidence on a negative test result. A variety of factors may contribute towards differing sensitivities of the test, such as patients age and level of parasitemia, which will vary according to endemic nature of P. falciparum in the locality. Lower test sensitivity may be related to low parasitemia in adults in an area of stable transmission. This may be a limitation of the tests; although, such patients are less at risk from severe clinical episodes to perpetuate parasite transmission.

Functions of Dehydrogenases in Health and Disease 177

Methodology. The methodology involved the use of patients both symptomatic and asymptomatic , who are villagers referred to the study and informed consent obtained. For each patient, a finger prick was made and the following were obtained. Fifty microliters of blood in a pre-heparinized Eppendorf tube for dipstick assays and two thick/ thin smears, one for on –site microscopic diagnosis by Acridine orange(AO) technique (for immediate treatment purposes, and the other, for reference Geimsa-microscopist.OptiMAL reader and ICT reader, were all blinded to each other's diagnosis. Patients positive for malaria were treated. Thick and thin blood smears were prepared and stained with Geimsa according to standard procedures. To declare a sample negative, thick smears were read for 200 microscopic fields (1000 X) without finding a parasite.If found positive, the number of asexual malaria parasites were counted per 500 WBC separately for each species. If there are more than 250 parasites/500 WBC, parasites were counted on the corresponding thin film per 10,000RBC. Density calculations were based on approximations of7500 WBC/µl and 5X10 6 RBC/µl. ICT malaria P.f/P.v test kits were used as per manufacturers instructions. Ten microliters (10µl) of whol blood was transferred to a sample pad. A buffer reagent was added to induce cell lysis and allow PfHRP 2 and pan malarial antigens to bind to colloidal gold-labeled antibodies. Additional buffer caused the blood and immune complexes to migrate up the test strip and cross monoclonal (mAb) lines. Finally, more buffer was added

Tests are counted as valid, if control lines are observed. They were counted P. falciparum positive, if Pf HRP 2 specific and pan-malarial antigen lines were visible or if only PfHRP 2 specific lines were seen only. If the control and pan-malarial antigen lines were observed, the sample was counted as positive for a malaria parasite other than P. falciparum. The test result was assigned a value of +0, if no line was seen; +1, if test line intensity was less than control line intensity; +2,if it was equal and +3, if it was greater in intensity. Other factors can exacerbate drug resistance. The second type of RDT detects the malarial antigen parasitelactate dehydrogenase (pLDH), an enzyme produced in the glycolytic pathway or cycle of the asexual stage of all species of plasmodium. Parasite lactate dehydrogenase is produced only by viable parasites, being cleared from the blood stream more quickly after treatment,

**9. Clinical and epidemological findings based on histopathology and immunohistochromatographic detection of p.falciparum antigens** 

This new technique for determination of P.falciparum is not adopted during autopsy when actual diagnosis of P.falciparum infestation is missed mostly during improper diagnosis. Many organs and tissues manifest the severity of this type of malaria before death. The heart, lung, liver tissues are always available for post mortem analysis. Some other tissues used include: spleen, kidney, and brain. The following tissues were equally used in a case of five travelers suspected to have died from other chronic diseases not related to malaria. These other tissues include: tongue, trachea, thyroid and adrenal glands, gall bladder and testis. Viral and or bacterial hemorrhagic fever pathogens were suspected at death [23, 24,

to clear blood from the membrane and facilitate reading.

resulting in the test becoming negative more quickly [22].
