Rrna molecules are polyadenylated



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1249
CONSIDERABLE PROPORTION OF LEISHMANIA BRAZILIENSIS 
RRNA MOLECULES ARE POLYADENYLATED
Marlene Jara Portocarreo, Jorge Arevalo
Instituto de Medicina Tropical Alexander von Humboldt, Lima, Peru
Leishmania parasiteare ancestral eukaryotes with unusual characteristics 
like polycistronic transcription and RNA trans-splicing. Like other 
eukaryotes, their RNA ribosomal genes are tandemly repeated and 
transcribed by RNA polymerase I. Unlike other eukaryotes, Leishmania 
ribosomes have rRNA molecules of 18S, 5.8S, and 28S, with the latter 
one being split into six rRNAs (
α.γ, β, δ, ζ and ε). The polyadenylation 
is a post-transcriptional process well known for mRNA but scarcely 
reported for rRNA. Our previous work on L. braziliensis and L. donovani 
demonstrated that at least the rRNA 28S 
ε undergo the polyadenylation 
process and that its relative abundance varies in Leishmania promastigote 
and amastigote stages. To determine if all rRNA gene subunits are 
subjected to polyadenylation, we evaluated the 18S rRNA, 5.8S rRNA and 
all the subunits homolog to 28S rRNA at stationary and logarithmic phase 
promastigotes of the L. braziliensis strain MHOM/BR/75/M2904. We found 
that all the rRNA subunits were polyadenylated. Moreover, we quantified 
the absolute amount of polyadenylated and non-polyadenylated rRNA 
of the sub-units 18S, 5.8S and 28S 
α by Reverse Transcription-Real time 
quantitative PCR. In the logarithmic promastigotes, the percentage of 
polyadenylated rRNA 18S, rRNA 5.8S and rRNA 28S 
α were 0.378 ± 0.02 
(mean ± standard deviation), 4.55 ± 0.43 and 13.86 ± 0.95, respectively. 
The stationary promastigotes had higher percentages of polyadenylated 
rRNA 18S (0.704 ±0.29, P=0.064) and rRNA 5.8S (5.69 ± 0.28, P=0.045) 
than the logarithmic promastigotes, whereas the 28S 
α did not show any 
significant differences between log and stationary promastigotes. These 
findings confirm a remarkable fact of Leishmania rRNA gene expression 
(also present in L.amazonensis, data not shown) and it is related to the 
parasite growth. The biological role of this phenomenon remains unknown 
but its wide conservation in the genus Leishmania indicates it is an 
important one.
1250
PHENOTYPIC CHARACTERISTICS OF ACUTE CHAGASIC 
MYOCARDITIS AMONG C57 AND BALB/C MICE
Andrés F. Henao-Martínez, Anne H. Agler, Timothy A. 
McKinsey, David A. Schwartz, Ivana V. Yang
University of Colorado Denver, Aurora, CO, United States
Chagasic disease is a notable neglected tropical disease with high 
morbidity in Latin America and among immigrants to the US. The primary 
mechanism of mortality is cardiomyopathy and sudden death. Acute 
chagasic myocarditis is consistently found in acute infections but little is 
known about its contribution to chronic forms of cardiomyopathy and 
what host factors play a role in acute myocarditis. The aim of this study 
was to phenotypically characterize two strains of mice with differential 
susceptibility to acute chagasic infection and correlate strain phenotypes 
with heart tissue gene expression. Laboratory mouse Tulahuen strain of 
Trypanosoma cruzi was grown in 3T3 fibroblast cell culture and tissue-
derived trypomastigotes (TCT) were harvested from supernatant. C57 
and Balb/c mice were injected intraperiotneally with 0 or 150-200 TCT. 
Weekly, mice were weighed and parasitemia was monitored via retro-
orbital blood sample. At 4 weeks Brain natriuretic peptide (BNP) and 
Troponin were measured in plasma and echocardiograms were obtained. 
4-week mortality was 56.3% and 12.5% for Balb/c and C57 (p=0.009), 
respectively. Infected Balb/c mice lost more weight than infected C57 
mice (p=0.018). Parasitemia peaked at 2 weeks, but was not significantly 
different between strains due to high variation in counts: 500,781 ± 
866,464 (Balb/c) vs. 140,625 ± 280,606 (C57) parasites/ml (p=0.12). For 
infected mice, BNP and troponin levels were not significantly different 
between strains, but BNP differed from uninfected mice. Echocardiograms 
demonstrated differences in heart rate in BALB/c vs. C57 mice: 413 vs. 
476 bpm, (p=0.0001) and stroke volume: 31.9 ± 9.3 vs. 39.2 ± 5.5 µl 
(p=0.03); therefore in cardiac output: 13.1 ± 3.5 vs. 18.7 ± 3.2 µl/min 
(p=0.002). There are relevant susceptibility and hemodynamic differences 
between these strains of mice during acute chagasic infection. Further 
characterizations of heart tissue histopathology, immunohistochemistry 
and gene expression will investigate possible host factor determinants for 
acute chagasic myocarditis.
1250A
QUANTITATIVE KDNA ASSESSMENT DURING TREATMENT OF 
MUCOSAL LEISHMANIASIS AS A POTENTIAL BIOMARKER OF 
OUTCOME
Marlene Jara
1
, Braulio M. Valencia
1
, Milena Alba
1
, Vanessa Adaui
1

Jorge Arevalo
1
, Alejandro Llanos-Cuentas
1
Andrea K. Boggild
2
1
Universidad Peruana Cayetano Heredia, Lima, Peru, 
2
University of Toronto, 
Toronto, ON, Canada
Mucosal leishmaniasis (ML) is a disfiguring manifestation of infection with 
Leishmania (Viannia) spp. As there is no known biomarker of treatment 
outcome in ML, we evaluated the concentration of kinetoplast minicircle 
DNA (kDNA) by cytology brush quantitative PCR before, during, and 
after treatment of ML in Peruvian patients. ML lesions were sampled by 
cytology brushes for quantitative PCR at enrolment, days 14 and 21_28 
of therapy, and 3-, 6-, or 12-mos after treatment. Parasite concentration 
in tissue was correlated to demographic, clinical, and parasitologic 
factors. Twenty patients completed follow-up: 12 men and 8 women, 
with median age of 37 yrs (range 18_78 yrs). Fifteen patients were 
treated with sodium stibogluconate, and 5 with amphotericin B. Cure 
was achieved in 17 patients, while 2 patients failed multiple courses of 
therapy. Clinical outcome is unknown in 1 patient. Mean parasite load 
(PL) at enrolment was 85,614.8 ± 60,427.3 parasites per ug of tissue DNA 
(par/ug tDNA). Three patterns of quantifiable kDNA during therapy and 
follow-up emerged: pattern 1 (N=10) was characterized by a mean PL of 
170,867 ± 117,482.6 at enrolment, with sequential decline in PL during 
and after therapy until kDNA was undetectable. Pattern 2 (N=4) was 
characterized by mean PL of 566.4 ± 306.4 at enrolment, with clearance 
of detectable kDNA by D14 of treatment, followed by an increased PL by 
D21-28 of treatment to 80.4 ± 32.1 par/ug tDNA. Pattern 3 (N=6) was 
characterized by mean PL of 226.7 ± 116.1 at enrolment, with clearance 
of detectable kDNA during treatment, followed by increased PL by 
6-mos follow-up to 36.6 ± 13.1 par/ug tDNA. Both patients who failed 
treatment demonstrated Pattern 1. Patterns 2 and 3 were associated with 
granulomatous inflammation (p=0.02). Younger age (33.5 vs. 64 yrs, 
p=0.10) and shorter ML duration (20.5 vs. 48 mos, p=0.11) are potentially 
correlated to sequential clearance (pattern 1). Baseline PL, sex, exposure 
duration, lesion number, and ML location were not correlated to pattern of 
PL. We have demonstrated that the concentration of parasite kDNA in ML 
can be quantified by cytology brush sampling and quantitative PCR during 
and after treatment. Interim analysis demonstrates 3 distinct patterns 
of PL during and after treatment, which warrant further investigation. 
Granulomatous inflammation may predict rebound of PL during or after 
treatment, though the clinical significance of this rebound is presently 
unknown.
1251
COMPARISON OF TWO COMBINATION PARASITE LACTATE 
DEHYDROGENASE-BASED RAPID TESTS FOR THE DIAGNOSIS 
OF MALARIA DUE TO PLASMODIUM KNOWLESI AND OTHER 
PLASMODIUM SPECIES IN SABAH, MALAYSIA
Matthew J. Grigg
1
, T. William
1
, B. E. Barber
1
, U. Parameswaran
1

T. W. Yeo
2
, N. M. Anstey
2
1
Queen Elizabeth Hospital, Kota Kinabalu, Sabah, Malaysia, 
2
Menzies 
School of Health Research and Charles Darwin University, Darwin, Australia
Plasmodium knowlesi human infection has been reported throughout 
South-East Asia, and is the most common cause of severe malaria in parts 

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of Borneo. Microscopic misdiagnosis is common, and may impact prompt 
initiation of treatment shown to improve mortality outcomes. Previous 
studies have shown cross-reactivity of P. knowlesi with parasite lactate 
dehydrogenase monoclonal antibodies used to detect P. falciparum and 
P. vivax. Our initial evaluation of rapid diagnostic tests (RDTs) has not 
demonstrated sufficient sensitivity for P. knowlesi, and no specific antibody 
for P. knowlesi has been developed. At both tertiary and district referral 
sites in Sabah, Malaysia, we prospectively evaluated two combination RDTs 
for the diagnosis of uncomplicated and severe malaria. Firstly with a pan-
Plasmodium parasite lactate dehydrogenase (pan-pLDH) and P. falciparum 
specific parasite lactate dehyrogenase (PfLDH) RDT (Optimal-IT). Secondly 
with a non-P. falciparum pan-parasite lactate dehydrogenase (VOM), and 
P. falciparum histidine-rich protein-2 (HRP2) RDT (Carestart). Among 250 
patients hospitalised with PCR-confirmed P. knowlesiP. falciparum and P. 
vivax monoinfection, the pre-treatment sensitivity of the pan-pLDH test for 
each species was 36% (49/137; 95% confidence interval [CI] 28 to 44%), 
75% (63/84; CI 64 to 84), and 83% (24/29; CI 64 to 94) respectively. The 
PfLDH test sensitivities were 33% (45/137; CI 25 to 41), 77% (65/84; CI 
67 to 86) and 14% (4/29; CI 4 to 32) respectively. The VOM component 
was the most sensitive test for both uncomplicated (44%; 60/137; CI 
35 to 53) and severe (79%; 15/19; CI 54 to 94) P. knowlesi malaria but 
remained clinically insufficient. More sensitive RDTs or alternative molecular 
diagnostic tools are needed in areas of P. knowlesi endemicity.
1252
COMPARATIVE ANALYSIS OF MALARIA INFECTIONS BY 
NESTED PCR USING A POOLING STRATEGY ON DRIED BLOOD 
SPOTS AND PLACENTAL HISTOLOGY IN MICROSCOPY-
NEGATIVE MALAWIAN WOMEN ON IPTP
Zhiyong Zhou
1
, Julie R. Gutman
1
, Dyson Mwandama
2
, Doreen 
Ali
3
, Don Mathanga
2
, Jacek Skarbinski
1
, Ya Ping Shi
1
1
Centers for Disease Control and Prevention, Atlanta, GA, United States, 
2
University of Malawi College of Medicine, Blantyre, Malawi, 
3
National 
Malaria Control Program, Ministry of Health, Lilongwe, Malawi
Malaria infection in pregnant women on intermittent preventive treatment 
in pregnancy (IPTp) often presents low parasite densities at delivery and 
this poses a great diagnostic challenge. In this study, a nested polymerase 
chain reaction (nPCR) assay for the 18S rRNA gene of Plasmodium 
falciparum was conducted to detect malaria infection in microscopy-
negative pregnant women at delivery from an IPTp effectiveness study 
conducted in Malawi. A sample pooling strategy was developed for 
screening malaria infection using dried blood spots samples (DBSs) 
collected from placenta or periphery at delivery. Considering a known 
malaria prevalence of 7.6% by microscopy in pregnant women at delivery, 
histologic results were used to stratify the 619 available microscopy-
negative samples into sample pools. Each sample pool contained 4 DBSs 
from histology-positive samples or 10 DBSs from histology-negative 
samples prior to DNA extraction for first round of nPCR screening. For 
those nPCR-positive pools, DBSs were then individually extracted and a 
second round of nPCR assay was performed. Overall, of 619 microscopy-
negative DBSs, 179 (28.9%) were positive by histology and 52 (8.4%) 
were positive by nPCR. Among the histology-positive samples, 39 (21.8%) 
had active infection (acute and chronic) and 140 (78.2%) had past 
infection. Using the histology results as a reference, 71.8% women were 
nPCR-positive in the active infection group, 7.1% were nPCR-positive 
in the past infection group, and 3.2% were nPCR-positive in histology-
negative group. In conclusion, histology diagnosis detected more malaria 
infection, but nPCR combined with a proper sample pooling strategy is 
still a practical and sensitive method to detect low density, active malaria 
infection at delivery. This study has demonstrated that nPCR can be a 
useful tool to detect submicroscopic malaria infection in pregnant women 
at delivery when histology diagnosis is not available.
1253
NATIONALLY REPRESENTATIVE SURVEYS OF MALARIA 
DIAGNOSTIC CAPACITY IN THE PUBLIC SECTOR: FINDINGS 
FROM GHANA AND BENIN
Joseph Keating
1
, Tim Finn
1
, Luis Benavente
2
, Chris Petruccelli
2

Nicole Whitehurst
2
, Thomas Eisele
1
, Gilbert Dery
3
, Ekow Biney
4

Benjamin Fayomi
5
Joshua O. Yukich
1
1
Tulane University School of Public Health and Tropical Medicine, New 
Orleans, LA, United States, 
2
Medical Care Development International, 
Silver Spring, MD, United States, 
3
DERMED Consult LLC, Tamale, Ghana, 
4
Ghana Health Services, Accra, Ghana, 
5
Insitut d’Sciences Biomedicales 
Appliquees (ISBA), Cotonou, Benin
In many African settings, malaria cases are treated presumptively. The 
absence of parasitological confirmation of malaria infection can lead to 
overtreatment of febrile illness with anti-malarial drugs, or the missing of 
other potentially fatal conditions. The development of rapid diagnostic 
tests for malaria (RDTs) combined with the scale up of Artemisinin 
Combination Therapies (ACTs) has led to increasing pressure to scale 
up parasitological diagnosis. In order to assess the availability, quality 
and accuracy of malaria diagnosis in Ghana and Benin, nationally 
representative health facility surveys were conducted in publicly supported 
health facilities in both countries. Results indicate that diagnostics are 
performed accurately a majority of the time when they are applied. The 
sensitivity and specificity of microscopy compared to expert readings was 
approximately 80% across all sampled facilities on the day of the survey. 
Furthermore, all observed RDTs in Ghana were interpreted correctly based 
on a surveyor’s re-interpretation. These results appear significantly better 
than historic literature on malaria diagnosis with microscopy in many 
African locations. In Ghana, in the majority of cases, clinicians gave or 
prescribed drugs in line with test results. While this result is promising, 
it only reflects practice among patients where a test result was received. 
Many patients were diagnosed with malaria clinically (i.e. in the absence 
of any test results). While few of the patients with negative test results 
received a malaria diagnosis, only half of all fever patients were referred 
for a malaria test. Despite the overall appearance of the acceptance of 
testing and the agreement of clinical prescribing practice with laboratory 
results, approximately 30% of patients who received negative test results 
still received anti-malarial drugs.
1254
MAGNETIC DETECTION OF HEMOZOIN SURPASSES GOLD 
STANDARDS OF MALARIA DIAGNOSIS AND RIVALS 
SENSITIVITY OF MOLECULAR BASED METHODS
John R. Lewandowski
1
, William C. Condit
1
, Robert J. Deissler
1

Richard F. Bihary
1
, Mark R. Lewandowski
1
, Jason E. Jones
1
, D’Arbra 
R. Blankenship
1
, Melinda J. Zikursh
1
, Arsene Ratsimbasoa
2
, Moses 
J. Bockarie
1
, Peter A. Zimmerman
1
, Robert W. Brown
1
, Brian T. 
Grimberg
1
1
Case Western Reserve University, Cleveland, OH, United States, 
2
National 
Malaria Control Programme, Antananarivo, Madagascar
Malaria parasites digest hemoglobin and in the process release cationic 
alpha-hematin, which is toxic to the red blood cell (RBC) and developing 
parasite. The developing parasite polymerizes this substance into 
chemically inert crystals known as hemozoin. Here we have exploited 
the paramagnetic properties of hemozoin to develop magneto-optical 
diagnosis (MOD) of malaria. When mixed with water, parasitized RBCs 
swell, burst open and release hemozoin into solution. Exposure of this 
lysate to an alternating magnetic field periodically aligns the hemozoin 
crystals so they block the transmission of light through the solution in 
proportion to parasitemia. When testing MOD on 291 samples from a 
malaria-endemic area we detected as few as 39 parasitized cells/µL from 
patients in less than1 minute with an overall accuracy of 93% compared 
to PCR based detection methods. Additionally, a subset of these patient 
samples were also compared to RDT (CareStart HRP2/pLDH (Pf/PAN) 

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COMBO) based detection methods which showed only 29% accuracy 
when compared to PCR results. Further studies of cultured parasites 
showed even lower detection of <1 parasitized cells/µL This device 
provides a rapid, robust, and inexpensive diagnosis of malaria which is an 
improvement over microscopic and RDT based diagnosis and allows for 
screenings on a population-based scale which is in line with the goals of 
global malaria elimination.
1255
SCALING-UP MALARIA RAPID DIAGNOSTIC TESTS AND 
ARTEMISININ-BASED COMBINATION THERAPY INTO 
INTEGRATED COMMUNITY CASE MANAGEMENT SITES: 
RESULTS FROM TWO REMOTE AND LOW-RESOURCE 
SETTINGS IN THE DEMOCRATIC REPUBLIC OF CONGO
John Otshudiema
1
, Narcisse Embeke
2
, Filiberto Hernandez
3
, Jose 
Tchofa
1
, Clarisse Mbo Modiri
4
, François-Xavier Mwema
4
1
United States Agency for International Development, Kinshasa, 
Democratic Republic of the Congo
2
Management Sciences for Health, 
Kinshasa, Democratic Republic of the Congo, 
3
Centers for Disease Control 
and Prevention, Kinshasa, Democratic Republic of the Congo, 
4
National 
Malaria Control Program, Kinshasa, Democratic Republic of the Congo
Integrated Case Management of Childhood Illness (iCCM) improves 
access to prompt, accurate diagnosis and effective treatment of malaria 
for populations with limited access to health facilities. In the Democratic 
Republic of Congo (DRC), a pilot study in 2008-2009 demonstrated the 
feasibility and the acceptability of integrated use of rapid diagnostic tests 
(RDTs) for malaria and artemisinin-based combination therapy (ACT) in 
remote villages by community health workers (CHWs). Scaling-up of the 
newly adopted strategy began in 2012, reaching currently 129 iCCM sites. 
This abstract reports the results of the scaling-up in two targeted sites 
in order to improve their implementation. Patients’ forms filed by CHWs 
from two targeted iCCM sites in Kanda-Kanda health zone were reviewed 
to assess their adherence to the new malaria treatment guidelines. From 
July 2012 through March 2013, 644 sick children under five years were 
managed by CHWs, out of which 432 (67%) were complaining of fever 
for less than two days without signs of danger. RDTs were performed on 
181 (42% of those with fever) children. The remaining uncomplicated 
cases were treated presumptively with ACTs. CHWs referred 12 severe 
cases to health facilities for proper case management. Among those 
tested, 169 (93%) had a positive RDT of which 166 (98%) were treated 
with ACTs. However, 11 of the 12 patients with negative RDTs were 
treated also with ACTs. Among those confirmed uncomplicated RDT-
positive cases treated with ACTs, 68% were treated within 48 hours of 
the onset of fever. iCCM has the potential to improve access to prompt, 
effective management of uncomplicated malaria in remote, low resource 
settings but challenges remain to improve CHW use of RDTs for diagnosis 
and adherence to test results.
1256
USING OUTCOME-DRIVEN INNOVATION THEORY TO CLARIFY 
TARGET PRODUCT PROFILES FOR NEXT-GENERATION 
MALARIA DIAGNOSTICS
Kathleen Tietje, Christine Clerk, Kenneth Hawkins, Sarah 
McGray, Paul LaBarre
PATH, Seattle, WA, United States
Diagnostic tools used to reduce the burden of malaria in the control 
phase are less effective in regions undergoing programmatic reorientation 
toward malaria elimination. Next generation diagnostics for malaria 
elimination will need to be more accurate than microscopy and existing 
rapid diagnostic tests to detect the reservoirs of low-density, asymptomatic 
infections that perpetuate disease transmission. In addition, new 
diagnostics will need to be user friendly, field deployable, and capable 
of high throughput at low cost. Despite ongoing progress in several 
diagnostic development programs, the technical and market requirements 
for elimination phase diagnostics remain ambiguous, and therefore 
developers lack the incentive necessary to bring new technologies to 
market. To address the need for detailed target product profiles (TPPs) for 
elimination-specific diagnostics, PATH’s project DIAMETER (diagnostics 
for malaria elimination toward eradication) team has identified a 
comprehensive list of outcome-based use-scenarios that are critical to the 
elimination context. Through a review of the literature and stakeholder 
interviews, we capture the essential system components, performance 
criteria, and market requirements that define success for malaria 
elimination stakeholders including health workers, global and national 
policymakers, and public and private health providers. Our findings will 
inform recommendations and TPPs to provide clear guidance ensuring the 
most efficient new diagnostic innovations are accelerated to market to 
support elimination campaigns.

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