Follow-up of HIV western blot indeterminate results.

Indeterminate results obtained with the Western blot (WB) confirmatory test on HIV enzyme immunoassay (EIA)-positive samples, constituted 15.6% (444/2849) over a 2.5-year period at the referral laboratory for the Western region at the King Fahd General Hospital, Jeddah. Two hundred and fourteen WB-indeterminate samples were followed up by repeat WB testing of subsequent samples from the same patients over a 3-12 month period. One hundred and forty-two samples (66.4%) gave negative results. Sixty-five samples (30%) remained indeterminate. Only seven samples (3.3%) not initially meeting WHO criteria for positivity turned clear-cut positive, with high EIA readings on follow-up. It was discovered initally that a significant proportion of indeterminates was due to low-grade cross-contamination between samples as a result of aerosol backflow during aspiration in the washing procedure. This was eliminated by rinsing the lines between samples, separating samples with high EIA from those with low EIA, and rerunning indeterminate samples. A reduction of indeterminates from 21% to 8.5% subsequently followed. After this improvement, most of the samples that remained indeterminate had low EIA readings, and few bands of mainly anti-gag (p55, p24 or p18) or anti-pol (p51) antibodies, while the few turning positive all had anti-gp160, in addition to anti-p24 or p55. Interestingly, over the last year and a half of the study, 1.4% of the total samples (21/1506) had repeatedly high EIA readings but were negative by WB. In addition, 16 samples (1.1%) were positive for HIV-2. A separate computer-based system for the storage of data was very helpful in ascertaining proper follow-up of indeterminate WB results.

Malaria in children - experience from Asir region, Saudi Arabia.

During a five year period, 233 cases of malaria (2.4%) were diagnosed among 9259 children with fever and hepatosplenomegaly seen in Asir Central Hospital, Abha, Saudi Arabia. The majority of these were below four years of age and came from Tihama, a hot, humid valley area in the Asir region. The infection was seasonal and occurred between December and May. Apart from fever, vomiting and hepatosplenomegaly, anemia was a common clinical finding; this was partly due to iron deficiency anemia, probably nutritional. Most of the cases responded to chloroquine therapy; however, three required intravenous quinine and two received Fansidar to effect eradication of the parasitemia. During the study, two patients died, one from cerebral malaria and the other from severe hemolytic anemia and hemoglobinuria. For prevention of malaria in this endemic area, an integrated program is advocated that includes the use of bednets impregnated with permethin, adequate treatment of proven cases and intensive health education on malaria control and nutrition.

Acceptability and usage of permethrin-impregnated mosquito bed nets in rural southwestern Saudi Arabia.

In 1989 a total of 2,320 sets of locally-made durable permethrin-impregnated bed nets and support frames were provided for the first time to 410 families (2,485 individuals) in Al-Fateeha area in the malarious region of Tihamat Asir, southwestern Saudi Arabia. In interviews carried out with a sample of the heads of the families two years later, most of them said to have used bed nets regularly (78.3%) and a majority started using them shortly after sunset (73.9%). Most heads of families expressed willingness to encourage their friends and neighbours to get bed nets (94.4%) and use them regularly (92.9%). Public requests for bed nets were received from neighbouring areas. These results indicate that impregnated bed nets can be successfully integrated into a malaria control programme provided that they are of a durable type and accompanied with support devices to facilitate their use while sleeping outdoors. Records of the primary health care centre serving the trial area indicated that the incidence of malaria decreased progressively from 277.4 per thousand in 1988 to 124.4 in 1991. The possible contribution of mosquito nets--as a major newly introduced variable--to this decrease is suggested but was not directly measured.

Respiratory syncytial virus infection in young children hospitalized with respiratory illness in Riyadh.

The occurrence of respiratory syncytial virus (RSV) infection among young children hospitalized with lower respiratory tract illness, at King Khalid University Hospital in Riyadh, was examined during the autumn-winter season between September 1991 and February 1992. Sixty-nine cases were diagnosed by immunofluorescent antibody staining of viral antigen in nasopharyngeal aspirates from 127 children, constituting 54 per cent of these patients. Virus culture was attempted only in a few cases, yielding two isolates. Most children were < 1 year of age (median 2 months). Bronchiolitis and bronchopneumonia were the major diagnoses on admission. Hospitalization was for an average of 5 days (range 1-36 days). Treatment was supportive but most children received antibiotic therapy. There was no mortality. Few other bacterial or viral pathogens could be identified from RSV-positive or -negative patients. These results indicate that, during the season of infection, RSV may be the main pathogen of lower respiratory tract illness in hospitalized young children in this region.

Serodiagnosis of hepatitis C in acute and chronic liver disease in southwestern Saudi Arabia.

The extent of involvement of hepatitis C, as compared to hepatitis A and hepatitis B, virus infection in acute and chronic liver disease in the Asir Region, southwestern Saudi Arabia, was assessed in 898 patients hospitalized during the period from June 1990 to November 1991. Acute icteric hepatitis cases with severe onset were distinguished by their referral to the fever hospital while cases with milder onset and those with chronic hepatitis were followed at two general hospitals. Antibodies to the c-100-3 antigen of hepatitis C virus (anti HCV) were detected in a significant proportion of patients with chronic liver disease (chronic active hepatitis (65%), cirrhosis (44%)). Anti HCV was also detected in patients with acute hepatitis with milder onset at the general hospitals (10.9%) but proportionately much less in patients at the fever referral hospital (< 1%) where hepatitis A (52%) and, to a lesser extent hepatitis B (11%), were mostly diagnosed. These results indicate that HCV is a major identifiable infection in hospitalized patients with chronic liver disease in this region but that anti HCV antibodies (c-100-3) are not detected, at least at onset, in sporadic cases with acute manifestations. Testing for additional viral antigens or RNA and a longer follow-up period would be required before exclusion of a role for HCV in acute disease. Alternatively, other viral and non-viral agents may be sought in this illness.

Fatal lipoid pneumonia in an infant complicated by Mycobacterium fortuitum infection.

We report a fatal case of lipoid pneumonia in a two and one half month old Saudi female caused by aspiration of animal fat, ghee and complicated by Mycobacterium fortuitum infection. It highlights the need to look for M. fortuitum in lipoid pneumonia and suggests the prevention of the condition through health education.

Protease inhibitors as potential therapeutic agents for AIDS.

A decade since the epidemic of the acquired immunodeficiency syndrome (AIDS) was first recognized, a wealth of information has accumulated on the molecular biology of the causative agents, the human immunodeficiency viruses (HIV). Of particular interest is knowledge of the viral enzymes involved in the formation of new virus particles. Such enzymes constitute attractive targets for efforts aimed at selecting agents that interfere with virus multiplication and subsequent spread and pathogenesis. Already, several agents that inhibit the viral reverse transcriptase (e.g., nucleoside analogs such as Zidovudine) have proved to have a beneficial effect on the course off the disease, but their prolonged use has been associated with significant toxicity and the emergence of resistant mutants. A second enzyme that has recently attracted attention is the virus-coded protease. This enzyme is involved in the cleavage of viral precursor polyproteins into the final products that constitute the mature virus particle. Protease inhibitors interfere with the process of virus maturation which is required for the formation of infective virus particles. Several custom-made inhibitors with a high selective action against HIV protease have been produced recently. They are nonhydrolyzable peptide analogs that mimic the cleavage sequences of the natural substrate of the enzyme during the transition state of the cleavage reaction. It is hoped that a similar selectivity in vivo may make protease inhibitors a promising new category of AIDS therapeutics.

A study of the knowledge, attitude and practice (KAP) of a Saudi Arabian community towards the problem of brucellosis.

The present study included 337 patients, presenting to Asir Central Hospital with fever of more than two weeks duration, or symptoms associated with brucellosis but without fever. Of the 337 subjects examined for knowledge about methods and means of transmission of brucellosis, 309 (92%) were ignorant while only 28 (8%) appeared to possess some knowledge as to the source, type of animal contact and presentation of illness. None of the 337 subjects was able to link the disease with a microbial infection. The most important common practices associated with brucellosis included raw milk consumption, close animal contact and the slaughtering and disposal of wastes. Illiteracy, ignorance and faulty behaviours emphasize the importance of health education of the community, to raise the KAP standard of the full spectrum of brucellosis in the community would be valuable in its prevention and control.

Portable mosquito net support devices for indoor and outdoor use.

Polyvinyl chloride plumbing and electrical cover pipes are used in the assembly of low priced devices that will support mosquito bed nets. One such device is a frame assembled from components in a lightweight compact set with dimensions of 75 x 20 x 5 cm, weighing approximately 0.5 kg. It can be mounted on a wall or attached to a stand of adjustable height which is also made from plumbing pipes. This support frame provides minimum obstruction of view and movement as it is attached at only one point. It can be used indoors or outdoors either for sleeping on beds or on the ground. A second device is composed of short pipe sheaths attached to bed legs for easy insertion or removal of support poles.

Formation and role of malaria pigment.

Malaria pigment is most abundant and distinct in gametocytes. Trophozoites have variable amounts of pigment, depending on the species of Plasmodium and the stage of infection. In Plasmodium falciparum infection, blood smear preparations fall into two categories that are distinguishable at all levels of parasitemia; one type of preparation contains only pigment-deficient trophozoites, and the other type contains only pigment-rich trophozoites. Pigment accumulates in the residual body of the mature schizont and is lost upon rupture of the schizont. In contrast, pigment remains associated with the macrogametocyte and developing oocyst. Certain antimalarial drugs, such as chloroquine, have distinct effects on pigment clumping. These observations raise questions regarding the current idea of pigment as an inert excretory product of hemoglobin metabolism. It is suggested that pigment particles represent stacked utilizable intermediates of hemoglobin digestion that accumulate in the gametocyte to serve as a food reserve during the growth cycle in the mosquito.

Patterns of pigment accumulation in Plasmodium falciparum trophozoites in peripheral blood samples.

Ninety-five samples of peripheral blood from patients with Plasmodium falciparum malaria in southwest Saudi Arabia were examined by Giemsa staining and darkfield microscopy under flow condition. Eighty-four samples contained trophozoites (ring forms) only and 11 samples contained gametocytes and trophozoites. Two patterns of pigmentation were observed in the trophozoite-containing samples: 48 (57%) contained trophozoites in which no pigment could be detected, 32 (38%) contained trophozoites with clearly detectable pigment, and 4 (5%) contained both pigmented and nonpigmented forms. Trophozoite pigmentation did not correlate with percent parasitemia or age or sex of the patients. These results indicate that microscopically observable pigment accumulation in trophozoites of P. falciparum is not required during the asexual multiplication cycle. Pigment accumulation may be triggered later in infection, perhaps as a feature of the differentiation process leading to the formation of gametocytes.

Dark-field microscopy for detection of malaria in unstained blood films.

Pigment present in different forms of Plasmodium species infecting humans exhibits light scattering when blood films are viewed by dark-field microscopy. This facilitates the detection of parasite-infected cells owing to their brightly illuminated appearance. The technique was described long ago for the detection of certain highly pigmented forms of malarial parasites such as schizonts and gametocytes but has not found an application in routine diagnosis. Here, modifications are described which allow the detection of all forms of human malarial parasites, including ring forms, in unstained blood films. The technique offers the distinct advantages of rapid diagnosis, increased sensitivity, and adaptability to field work.

Brucellosis in Riyadh, Saudi Arabia. A microbiological and clinical study.

During a period of two years, 30 cases of brucellosis were positively diagnosed from a total of 209 patients who reported with prolonged fever for investigation. Diagnosis was made both by blood culture and serological tests. The latter included slide and tube agglutination in all cases and an enzyme linked immunosorbent assay (ELISA) in 16. 11 cases (36.7%) gave negative results by the slide-agglutination screening test used at the recommended single serum dilution of 1:80. This was due to the prozone phenomenon as they gave positive results upon further dilution in the tube agglutination test. 13 of the 16 tested by ELISA were positive for both IgM and IgG and three were positive for IgG only. Of the six cases that were positive by culture, five grew Brucella melitensis and one B. abortus.

Biosynthesis of reverse transcriptase from Rauscher murine leukemia virus by synthesis and cleavage of a gag-pol read-through viral precursor polyprotein.

Reverse transcriptase (RT; RNA-dependent DNA nucleotidyltransferase) from Rauscher leukemia virus is synthesized in infected cells by way of a read-through poly- rotein of 200,000 molecular weight. This polyprotein (Pr200(gag-pol)) was precipitated by antiserum to RT; in a previous study all the monospecific antisera to gag proteins recognized Pr200(gag-pol). Pr200(gag-pol) contains both p30 and RT peptide sequences. Intermediate RT-related precursors of 145,000 (Pr145(pol)), 135,000 (Pr135(pol)), and 125,000 (Pr125(pol)) molecular weights were specifically recognized by precipitation from infected cell extracts by antiserum to RT. These proteins shared methionine-containing tryptic peptide sequences with a virion polypeptide of 80,000 molecular weight (p80(pol)) precipitate by antiserum to RT. Purification of active RT enzyme from virions labeled with [(3)H]methionine showed that p80(pol) was the major component, based on analysis by gel electrophoresis and tryptic peptide mapping experiments. A polypeptide (Pr80(pol)), similar in size to mature viral p80(pol), was also precipitated from infected cells by antiserum to RT. Its peptide map was nearly identical to that of virion p80(pol). Pulse-chase studies showed that Pr80(pol), Pr125(pol), and Pr135(pol) were stable polypeptides, whereas Pr200(gag-pol) and Pr145(pol) were unstable precursors. Pulse-chase studies with the protein synthesis inhibitor, cycloheximide, showed that the processing of Pr200(gag-pol) occurred for a short time in the absence of protein synthesis.

Inhibition of maturation of Rauscher leukemia virus by amino acid analogs.

Synthesis of primary precursor polyproteins of Rauscher leukemia virus (RLV) core and envelope proteins occurs in the presence of amino acid analogs canavanine and p-fluorophenylalanine, but cleavage of these precursors is severely inhibited or slowed down. After treatment with these agents, the release of characteristic virus or stable virus-like particles is greatly depressed.

Selective decrease in the rate of cleavage of an intracellular precursor to Rauscher leukemia virus p30 by treatment of infected cells with actinomycin D.

The cleavage of an intracellular 67,000- to 70,000-dalton precursor, termed Pr4 to Rauscher leukemia virus (RLV) p30 protein proceeded at a slower rate when virus-producing cells were treated with actinomycin D (AMD). Treatment with AMD also caused a slight accumulation of Pr4 in purified early virus particles produced by a cell line which usually produces virions that contain little Pr4. The cleavage of other intracellular viral precursor polypeptides was not affected by treatment with AMD. Treatment of infected cells with cycloheximide, on the other hand, allowed the cleavage of Pr4 to proceed at the usual rate for a short period of time before further cleavage was drastically slowed or prevented. The cleavage of several other viral precursor polypeptides was also inhibited by treatment with cycloheximide. Different lines of evidence suggest that the mechanism of action of AMD is not due to a possible indirect effect on protein synthesis. Thus, the rate of cleavage of Pr4 was not affected by the length of pretreatment with AMD between 1 to 8 h. In addition, the combined effect of AMD and cycloheximide, at their maximal inhibitory concentrations, was greater than the effect of either drug alone, indicating the involvement of two at least partially different mechanisms in the action of AMD and cycloheximide. Furthermore, AMD did not affect the pulse labeling of viral precursor polypeptides. These results suggest that the interaction with viral RNA, whose production is inhibited by AMD, accelerates the cleavage of Pr4 to p30 during virus assembly. A hypothetical model is presented to illustrate th possible advantages of having a step in virus assembly in which genomic RNA interacts with a precursor to capsid proteins before the cleavage of that precursor.

A fucose-deficient glycoprotein precursor to Rauscher leukemia virus gp69/71.

Rauscher leukemia virus glycoprotein gp69/71 is synthesized in virus-infected cells by way of a 90,000 dalton glycoprotein precursor, termed Pr2a+b. This precursor could be labeled with radioactive glucosamine and methionine but not with fucose; whereas gp69/71 could be detected by labeling with radioactive glucosamine, fucose, or a mixture of amino acids but seemed to be deficient in methionine relative to Pr2a+b. Pr2a+b and gp69/71, were specifically precipitated by an antiserum prepared against phosphocellulose purified Rauscher gp69/71. Other virus-specific precursors, in addition to Pr2a+b, could be precipitated by antiserum prepared against detergent disrupted virus. Neither Pr2a+b nor gp69/71 was precipitated from cell extracts by antisera to Rauscher p30. Tryptic maps of Pr2a+b and gp69/71 showed that these glycoproteins share many tryptic peptides. Pulse-chase experiments with 14C-labeled amino acids indicated that gp69/71 was not radio-labeled during the pulse-labeling period but slowly appeared during the chase incubations. Pr2a+b, however, was rapidly labeled and tended to disappear during long chases. Furthermore, two nonglycosylated viral proteins, termed p15E and p12E, are structurally related to Pr2a+b. Viral p15E and p12E contained the same methionine-containing tryptic peptide fraction as Pr2a+b as determined by ion-exchange chromatography. These results provide evidence that Pr2a+b is a precursor to gp69/71 and establish a structural and possible precursor-product relationship between Pr2a+b, p15E, and p12E.