Malaria Prevalence in a Low Transmission Area, Jazan District of Southwestern Saudi Arabia

Detailed description of malaria in low transmission areas is crucial for elimination. The current study aimed to provide a comprehensive description for malaria transmission in Jazan, a low transmission district, southwestern Saudi Arabia. Patients at a tertiary care hospital were recruited in our study between August 2016 and September 2018. Malaria diagnosis was performed through a species-specific nested polymerase chain reaction (nested PCR), microscopy and Paramax-3™ rapid detection test (RDT). Malaria was detected in 30 patients by the PCR, with point prevalence of 10.9%. Of these malaria infections, 80% was imported, 26.6% was asymptomatic and 23.3% was sub-microscopic. Malaria was reported throughout the year, with February/March and September/October peaks. Infection was significantly more in males than in females (P=0.01). Likewise, infections were detected more in febrile than in non-febrile patients (P=0.01). Adult aged 15–24 years, fever and travel were identified as high-risk factors. Malaria was primarily attributed to Plasmodium falciparum mono-infections, followed by P. vivax mono-infections and lastly to falciparum/vivax mixed infections accounting 76.6%, 16.6%, and 6.6% of PCR-confirmed malaria cases, respectively. The nested PCR was superior to the smear microscopy (sensitivity 76.6%; specificity 100%) and the RDT (sensitivity 83.3%, specificity 94.2%). The overall percent agreement between microscopy and the RDT was 92.7% (kappa=0.63). High proportion of imported malaria including sub-microscopic and sub-patent cases were described. We suggest that incorporation of molecular tool into the conventional malaria diagnosis is beneficial in Jazan district.

Internal Amplification Control for a Cryptosporidium Diagnostic PCR: Construction and Clinical Evaluation

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (approximately375 bp) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects approximately550 bp of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, approximately2 oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.

Prevalence of Cryptosporidium-Associated Diarrhea in a High Altitude-Community of Saudi Arabia Detected by Conventional and Molecular Methods

Cryptosporidium diarrhea represents a relevant clinical problem in developing countries. In Al-Taif, a city of Saudi Arabia that lies at an altitude of an around 2 km above the sea level, Cryptosporidium infection seems to be undiagnosed in nearly all clinical laboratories. Furthermore, nothing was published regarding Cryptosporidium-associated diarrhea in this area. The objectives of this research were to (1) determine the Cryptosporidium prevalence among patients with diarrhea and (2) to estimate the performances of 3 different diagnostic methods. Total 180 diarrheal fecal samples, 1 sample per patient, were collected between January and August 2013. Samples were screened for Cryptosporidium with modified Zeihl Neelsen (ZN) microscopy, RIDA(R) Quick lateral flow (LF) immunotest, and a previously published PCR. The Cryptosporidium prevalence rate was 9.4% (17/180), 10% (18/180), and 11.6% (21/180) by microscopy, LF, and PCR test, respectively. Infection was significantly (P=0.004) predominant among children <5 years (22%) followed by children 5-9 years (11.1%). Although infection was higher in males than in females (16.2% males and 8.5% females), the difference was not statistically significant (P=0.11). Compared to PCR, the sensitivity of microscopy and the LF test were 80.9%, 85.7%, respectively. To conclude, high Cryptosporidium-associated diarrhea was found in this area especially in children < or =9 years. The PCR test showed the best performance followed by the LF test and ZN staining microscopy. The primary health care providers in Al-Taif need to be aware of and do testing for this protozoon, particularly for children seen with diarrhea.

DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR

PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp(R) DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 microl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, approximately 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.