Gender Inuence on Asymptomatic Malaria Diagnosis

The characterisation and identication of malaria reservoirs are major challenges for the ecacy of control and elimination programs. Malaria asymptomatic infections are highly prevalent in different malaria settings worldwide and constitute a great obstacle for disease control and elimination. The monitoring of asymptomatic malaria infection requires active case detection in many cases challenging the limit of diagnostic technology detection. We evaluated diagnostic methods and analyzed the gender dimension implication for asymptomatic malaria diagnosis on a high-endemic malaria region during the dry-season.


Introduction
Malaria is declining worldwide since the beginning of this century due to massive investment and remarkable expansion in the range and effectiveness of products and strategies available for its elimination [1]. As more countries start entering pre-elimination phase, strategies for detecting and target all hotspots of infection, whether geographic or demographic, become extremely important to treat all infections and interrupt malaria transmission [2]. Asymptomatic malaria infections remain a challenge for malaria control programs as it signi cantly in uences transmission dynamics [3]. Therefore, understand malaria infections and its detection among asymptomatic groups is of crucial importance in the near future for malaria control worldwide.
Accurate detection of asymptomatic malaria infections provide better realistic estimations of malaria burden and improve malaria control interventions. Due to the inherent lack of clinical manifestations, common sub-patent level of parasites, different malaria parasite species, different malaria parasite cellular stages, and other multivariable factors, the parasitological detection of asymptomatic malaria infections is still a challenge.
The commonly observed low parasitaemia infections among asymptomatic cases constitute a silent reservoir for malaria transmission. In this context, present molecular polymerase chain reaction (PCR) based methods are important tools since it can achieve 100-fold greater sensitivity as compared to microscopy [4]. Inhere we compared the use of microscopy and PCR to detect asymptomatic malaria infections on a highly endemic African community.

Methods
We performed the golden standard microscopic malaria diagnosis in 57 malaria asymptomatic adults PCR was performed using a T100TMThermal Cycler (Bio-Rad) under the following conditions: 95ºC for 5 min, followed by 35 cycles of 94°C for 30s, 60°C for 30s, 72°C for 1 min and with extension step of 72°C for 5 min. The PCR product was con rmed by electrophoresis gel (1% agarose gel stained with GreenSafePremium (NZYTech)) and visualized using an UV transilluminator (Gel-DocTM EZ Imager (BioRad) and quickly cut to minimize the UV exposure. 100 ng/µL of PCR products and 10 µmol/L of each primer (described above) were sent for sequencing (STABvida).
The 430bp-fragment obtained by PCR was extracted and puri ed from the gel slice using GRS PCR & Gel Band Puri cation Kit (GRiSP) and insert in the pJET plasmid using CloneJET PCR Cloning Kit (Thermo Scienti c™). The plasmid was isolated from the cultured bacteria using the NZYMiniprep (NZYTech) according to the manufacturer's instructions. 100 ng of plasmid DNA and 10 µmol/L of each primer (pJET1.2-forward and pJET1.2-reverse) were sequencing (STABvida) and analyzed with Geneious software.

Results
Within the patients included, eighteen patients showed high body temperature (> 37.5ºC) which was not associated with malaria infections detected or gender (Fig. 1B). Overall, 35% (20/57) patients were diagnosed with malaria using at least one diagnostic method. The use of PCR signi cantly increased the number of malaria infections detect from 16% (9 infections by microscopy) to 35% (20 infections by PCR) (p-value = 0.03) Fig. 1C. All the 9 infections detected by microscopy were also detected by PCR con rming. The detection of additional 11 sub-microscopic infections by PCR increased malaria positivity in 55%.
Strikingly, the observed difference between microscopy and PCR diagnosis was determined by gender.
Among males, PCR diagnosis had a residual increase to 8 infections from initial 6 infections detected by microscopy. However, among females, a 4-fold increase was observed (7-29%, p-value = 0.02) Fig. 1D. No other variables were signi cantly different comparing gender groups.
To evaluate the interference of possibility Plasmodium spp. infections, we sequenced all positive PCR infections. Three out of the 20 positive infections showed suspected mixed infections of P. falciparum and P. malariae, being that the remaining malaria infections con rmed the presence of pure P. falciparum infections (85%, 90% CI 64%-94%). The PCR products with mixed Plasmodium spp. were cloned to isolate DNA chains to con rm the presence of P. falciparum and P. malariae (Genebank accession number: MW094305). No gender association with Plasmodium spp. mixed infections since only two P. malariae were found on the male group and one on the female.

Discussion
On this prospective study, a prevalence of 35% asymptomatic malaria cases is detectable among asymptomatic infections on this high-endemic region during dry-season. We were able to con rm the superior capacity of molecular tools to diagnose submicroscopic malaria infections, which accounted to a 55% increase for the total malaria cases detected. Interestingly, applying PCR diagnosis, we found that submicroscopic infections were more prevalent among females as compared with males. Several factors have been reported to in uence asymptomatic cases of malaria such as age, gender, ethnicity, seasonality and/or geography. In particular, submicroscopic infections in women are commonly observed during pregnancy [5]. On our study, none of the women reported to be pregnant. Comparison of asymptomatic malaria diagnosis by microscopy and PCR. A) Average age mean of the groups analyzed. B) Percentage of individual with body temperature above 37.5 ºC among the groups analyzed. C) Percentage (%) of malaria infections detected by microscopy and by PCR). D) Difference of malaria diagnosis strati ed by gender comparing microscopy and PCR. Asterix marks a Fisher´s exact test with a p-value below 0.05.