Molecular surveillance of Plasmodium falciparum chloroquine resistance transporter variant T76 in Jazan area, Kingdom of Saudi Arabia.

The development of chloroquine as an antimalarial drug and the subsequent evolution of drug resistant Plasmodium strains had major impacts on global public health in the 20th century. In P. falciparum, the cause of the most lethal human malaria, chloroquine resistance is linked to multiple mutations in PfCRT, a protein that likely functions as a transporter in the parasite's digestive vacuole membrane. Rapid diagnostic assays for PfCRT mutations are already employed as surveillance tools for drug resistance. However, several reports have been published demonstrating cases with CO resistance. Sporadic cases have been reported as well as one large scale study demonstrated 12.4% resistance. However, all these reports were based on treatment failure (in vivo). rather than in vitro or molecular bases. Evidence suggests a crucial role for a point mutation in the P. falciparum chloroquine resistance transporter (pfcrt) gene on chromosome 7 in conferring CQ resistance. The mutation in the K76 codon in 3 cases out of 60 (5%) using ApoI restriction enzyme was detected. Although the percentage of drug resistance was not quite disturbing, but represented the possible establishment of chloroquine-resistant P. falciparum in Saudi Arabia, or the beginning of resistant strains by labors coming from abroad. Cross-border importation of resistant strains from neighboring countries must be considered. In vivo tests must be conducted parallel with the molecular markers to estimate more precisely the actual prevalence of resistance. Validation of molecular markers is urgently required and needs strong collaborative partnerships between subregional and regional networks.

Latex agglutination and indirect immunofluorescence tests in the diagnosis of Toxoplasma gondii in Saudi Arabia.

Commercial latex agglutination (LA) was assessed for Toxoplasma antibody screening. The sensitivity and specificity were compared with the reference standard indirect immunofluorescence (IFA). A total of 186 sera were collected from May 2008-October 2008 for Toxoplasma antibody by LA & IFA. Antibody to T. gondii 51/186 (27.4%) were LA-positive and 42/186 (22.6%) were IFA-positive. The sensitivity, specificity and positive predictive value of LA were 100%, 93.7% & 82.3% respectively. The nine LA-false positive sera were examined for rheumatoid factor and antinuclear antibodies, but were negative and none exhibited nonspecific polar staining as a cause for the false positivity.

Comparison of two commercial assays and microscopy with PCR for diagnosis of malaria.

This study compared conventional PCR with microscopy and 2 rapid detection methods, the pLDH which detected lactic dehydrogenase enzyme produced by actively metabolizing organisms and the malaria antibody tests. The sensitivity of PCR was 1 parasite/microl, i.e.: 50 times more sensitive than microscopy. When PCR was compared with microscopy, the sensitivity and specificity were 90% & 100% respectively. The sensitivity recorded was pLDH test in comparison to PCR (95%). The malaria antibody test recorded the least sensitivity (68%) PCR proved as the gold standard for evaluation of applied tests and the newly introduced ones. In absence of an expert microscopist, the pLDH test could substitute for microscopy. The test proved valuable to assess clinical cure, and predict drug resistance. Its advantage over microscopy was the ability to diagnose infection with low parasitemic patients. Antibody rapid tests might be not valuable in acute cases, but still accepted as a tool in epidemiological studies and in screening patients in blood banks in malaria endemic areas.

Evaluation of a real-time polymerase chain reaction assay for the diagnosis of malaria in patients from Jazan area, Saudi Arabia.

A real-time PCR assay with conventional microscopy by Giemsa-stained blood films was used. PCR was completed in an hour and identified the Plasmodium species in a single reaction. Blood was collected, and DNA was extracted. A genus-specific primer set corresponding to 18S ribosomal RNA was used to amplify target sequence. Fluorescence resonance energy technology hybridization probes were designed for P. falciparum over a region containing base pair mismatches allowed Plasmodium species differentiation. Microscopically positive patients (n = 60) were positive with real-time assay (100% sensitivity). 58 were single-species infections caused by P. falciparum; mixed infections (P. falciparum & P. vivax) were shown by real-time assay. Six out of 30 negative microscopy specimens were positive by real-time PCR (80% specificity). The discrepant results could be due to the subjective nature of microscopy and analytical objectivity of PCR, and high analytical sensitivity of real-time assay (1 parasite/microl) compared to microscopy (50 parasites/microl). Six patients were retested with ICT malaria test and 4 were positive showing that PCR results were correct. There was low correlation between parasitemia by microscopy and gene copy number for P. falciparum (r = 0.2; P = 0.05 [Spearman]).

Anemia, interleukin-10, tumor necrosis factor alpha, and erythropoietin levels in children with acute, complicated and uncomplicated malignant malaria in Jazan, Saudi Arabia.

To gain insight into potential relationships between tumor necrosis factor alpha (TNF-alpha), interleukin 10 (IL-10), erythropoietin (EPO), and anemia in acute malaria, 90 children 3 to 11 years with acute malaria were studied. According to parasitemia and hemoglobin levels, they were divided into 3 groups: G1 (mild): asexual low-density Plasmodium falciparum parasitemia <8000 parasites/ul and hemoglobin levels >8g/dl. G2 (high-density uncomplicated): asexual high-density parasitemia (>8000 parasites/ul, with hemoglobin levels >8 g/dl. G3 (anemia): with severe malaria symptoms and parasitemia with anemia (hemoglobin levels <8 g/dl). Hospital controls included 10 children with matching age group who required inpatient management but had no malaria parasitemia. Good marrow response was in G1 & G2 showed by elevation of serum EPO and soluble transferring receptors (sTfR) and increased red cell distribution width (RDW). In G3, bone marrow suppression was in spite of increased EPO level in response to anemia. TNF-alpha level was significantly higher G2 and G3 (P.05). IL-10 levels in G1 were significantly higher than in hospital control group (P<0.05). The highest level of IL-10 was in G2. The mean IL-10 to TNF-alpha ratio in G2 (4.64) was significantly higher (P<.005) than in G3 (mean ratio, 1.77).

HCV-RNA and anti-HCV IgM in Egyptian subjects bearing IgG anti-HCV antibodies.

The hepatitis C virus (HCV) infection is associated with a wide spectrum of clinical entities ranging from asymptomatic carriage to severe forms of chronic hepatitis. In Egypt, HCV infection has been shown to be highly prevalent. The aim of this study was to determine the frequency and significance of anti-HCV IgM in the sera of clinically healthy blood donors and chronic HCV patients, whose sera were also positive for anti-HCV IgG. Anti-HCV IgM was detected in the sera of 7 (46%) of the blood donors (n = 15), of whom 5 (71%) had a positive HCV-RNA. The corresponding results in patients with a chronic hepatitis C (CHC) infection (n = 19) were 8 (42%) and 5 (62%) respectively. The detection of anti-HCV IgM did not correlate with a positive test for HCV-RNA (R = 0.2) in the CHC patients. However, the levels of anti-HCV IgM in CHC patients were associated significantly with the level of serum transaminase, a finding that can be used in monitoring disease activity in such a group of patients. On the other hand, a significant association was evident between the detection of anti-HCV IgM and HCV-RNA in the sera of blood donors. Thus among the blood donors, viraemia correlates well with the detection of HCV-IgM Ab, but it cannot be excluded in its absence. The presence of HCV-IgM in some patients with CHC infection indicates that the antibody as a viral marker may not be unique to acute HCV infection.

Nontypeable bacteriophage patterns of methicillin-resistant Staphylococcus aureus involved in a hospital outbreak.

Of 93 strains of Staphylococcus aureus isolated from inpatient wards of Ismailia General Hospital, 48 (51%) were proven to be methicillin resistant (MR). Of these MR S. aureus strains, 44 were isolated from patients and 4 were isolated from healthy carriers, who were newly arrived interns working in the same wards. Bacteriophage patterns of MR S. aureus were identified by using routine test dilution (RTD) and 100-fold dilutions (100 RTD) of phages. Of these 48 strains, 37 (75%) (33 from patients and 4 from interns) were nontypeable when using RTD and 100 RTD of phages. Of the other 11 strains, 8 were nontypeable by RTD of phages, but 5 of them had the phage pattern D11/1136 when tested by 100 RTD. Three strains had the phage pattern 3A/3C/55/71, and three strains had different phage patterns, 29/81, 96, and 95/D11. The finding of colonization with virulent MR S. aureus strains in interns working on the wards in which these patients were located suggested that new strategies for control of MR S. aureus nosocomial infections must be considered and evaluated.

Clonal nature of Salmonella typhi and its genetic relatedness to other salmonellae as shown by multilocus enzyme electrophoresis, and proposal of Salmonella bongori comb. nov.

Crude cell extracts of 26 isolates of Salmonella serotype typhi (S. typhi) and 48 other Salmonella isolates representing 28 serotypes and seven DNA hybridization subgroups were analyzed for electrophoretic variants of 24 metabolic enzymes by starch gel electrophoresis. All strains of S. typhi had identical isoenzyme patterns, indicating that they were a single clone. All of the enzymes detected in the remaining strains were polymorphic, and the degree of genetic variation was quite high. The average number of alleles per enzyme locus was 4.7, and the mean genetic diversity per locus was 0.556. Thirty-two distinct allele profiles, or electrophoretic types (ETs), were found in these 48 strains of Salmonella serotypes other than S. typhi. Analysis of the genetic relationships of the ETs to each other showed that, with one exception, the ETs formed subgroups that were consistent with the subgroupings based on DNA hybridization studies. ET profiles were not always linked to specific serologic patterns. These data show that multilocus enzyme electrophoresis has a potential application in epidemiologic and taxonomic studies of salmonellae, although it is not differential for S. typhi. We also propose a new species, Salmonella bongori comb. nov., a new combination base on the elevation of Salmonella choleraesuis subsp. bongori to the level of species.

A study of Salmonella typhi isolated in Suez Canal area. Biotyping, phage typing and colicinogenic property.

In this work a total of 82 strains of Salmonella typhi were isolated from Egyptian patients diagnosed as quiry enteric fever. These cases were from Ismalia, Suez and port Said Areas. The strains fell in 16 phage types. Phage types N, 40, E1, and degraded Vi were the commonest phage type in Ismailia, while phage types degraded Vi and C1 were the commonest in Port Said. Phage types Di-N, degraded Vi, A and C1 were the commonest in Suez. Chemotyping of Salmonella typhi showed that the majority of the strains belonged to chemotype I (82%), and the rest belonged to chemotype II (18%). Colicin production was negative and all the strains were susceptible to the currently used antibiotics.