High specificity of semi-nested multiplex PCR using dried blood spots on DNA Banking Card in comparison with frozen liquid blood for detection of Plasmodium falciparum and Plasmodium vivax.

BACKGROUND: Venipuncture sampling in test tubes for detecting malaria parasites using PCR assays possesses a number of limitations such as reluctance of patients, some difficulties in transportation of blood samples and freezing them for long time. To overcome the mentioned limitations, some approaches have been employed by a number of authors. This study was proposed to compare between DNA Banking Card (DBC) filter papers containing dried finger-prick blood and venipunctured frozen liquid blood. METHODS: A total of 75 specimens was prepared from the equal enrolled individuals using three blood storage approaches; making Geimsa-stained thin and thick smears from each individual to determine the malaria-positive or -negative specimens, spotting two to three drops of finger-prick blood onto the DBC filter paper, and collecting a 2-ml venous blood sample into EDTA-contained test tube from each individual. A semi-nested Multiplex PCR technique with DNA extracted from the two latter sets of specimens was used for plasmodia diagnosis. RESULTS: DNA samples isolated from dried blood spotted on the DBC filter papers resulted in 32 (42.7%) positive and 43 (57.3%) negative cases comparable with the results outcome of frozen liquid blood with 35 (46.7%) positive and 40 (53.3%) negative cases. Statistical analysis revealed higher sensitivity for SnM-PCR using DNA from liquid blood with 100% vs. dried blood spotted on DBC with 97% but higher specificity for the DBC with 100% vs. liquid blood with 95.2%. CONCLUSIONS: Based on the results obtained from this study to overcome the problems of venipuncture frozen liquid blood sampling, replacement of a reliable filter paper for preserving finger-prick blood samples is a trustable and useful facilitator particularly in remote malaria-endemic areas.

Seroepidemiological study of visceral leishmaniasis among humans and animal reservoirs in Bushehr province, Islamic Republic of Iran.

Using direct agglutination tests, a survey of visceral leishmaniasis was carried out among children and adults from 13 villages and from nomadic tribes in Bushehr province during 1998-99. Of the 1496 plasma samples, the overall seropositive rate (titres > or = 1:3200) was 3.4%. Almost all cases (94.1%) were in children under 10 years old. Eighteen patients were diagnosed with kala azar; fever and splenomegaly were the predominant signs and symptoms. Parasitology and serology examinations of local animals identified dogs and jackals infected with Leishmania infantum. Suggestions for control of visceral leishmaniasis in this area are to eliminate stray dogs, identify cases among humans and suspected leashed dogs, and treat infected individuals.

Seroepidemiological study of visceral leishmaniasis among humans and animal reservoirs in Bushehr province, Islamic Republic of Iran

Using direct agglutination tests, a survey of visceral leishmaniasis was carried out among children and adults from 13 villages and from nomadic tribes in Bushehr province during 1998-99. Of the 1496 plasma samples, the overall seropositive rate [titres > or = 1:3200] was 3.4%. Almost all cases [94.1%] were in children under 10 years old. Eighteen patients were diagnosed with kala azar; fever and splenomegaly were the predominant signs and symptoms. Parasitology and serology examinations of local animals identified dogs and jackals infected with Leishmania infantum. Suggestions for control of visceral leishmaniasis in this area are to eliminate stray dogs, identify cases among humans and suspected leashed dogs, and treat infected individuals

Cyfluthrin (EW 050)-impregnated bednets in a malaria control program in Ghassreghand (Baluchistan, Iran).

In a study carried out in the Ghassreghand Division (Baluchistan, Iran) from March through November 1995, efficacy of cyfluthrin-impregnated bednets was compared to that of untreated nets, in relation to malaria control. Ten villages with a total population of 4,572 and 3 villages with a total population of 1,935 were used as treatment and control, respectively. The collection, impregnation (target dosage of 40 mg active ingredient [AI]/m2), and redistribution of the nets (9% nylon, 52% light cotton, 30% medium cotton, and 9% heavy cotton), carried out in mid-April, were done by local health workers, supervised by the senior research staff. Anopheles culicifacies was considered to be the main vector of malaria in the named area. This species is mainly zoophilic, endophilic, and exophagic. The initial uptake of the insecticide was lower than the target dosage, with high variation (nylon, 12.5 +/- 5.4 mg AI/m2; light cotton, 33.3 +/- 26.1 mg AI/m2; medium cotton, 25.9 +/- 20 mg AI/m2; heavy cotton, 17.6 +/- 12.5 mg AI/m2). The use of impregnated mosquito nets (used primarily outside) had no significant effect on the incidence of malaria. No difference was detected in the parasite density of patients with positive slides. No significant effect was observed in the parous rate, human blood index, and sporozoite rate of anopheline vectors. Only the indoor resting densities of An. culicifacies and other malaria vectors were drastically reduced after the introduction of the cyfluthrin-impregnated nets into the treatment villages. The residual activity of cyfluthrin was lower than expected. The mortality of anophelines brought in contact with the treated nets for 3 min in bioassays dropped to less than 55% in 3 months. The loss of chemical activity was greatest for the light cotton nets, followed by the medium cotton nets. Cyfluthrin-treated nets were mildly irritating to host-seeking female anophelines in the laboratory. The protective rate of impregnation (all fabric kinds included) in preventing female mosquitoes from biting through the impregnated nets was initially 5-6 times that of the nonimpregnated nets. The study did not detect any significant difference between the use of untreated versus impregnated bednets in the Ghassreghand area. In planning future medium-scale trials, comparison of new compounds and formulations to the more widely used pyrethroids such as permethrin and deltamethrin is highly recommended.

Assessment of the response in vivo and in vitro of Plasmodium falciparum to sulphadoxine-pyrimethamine in the malarious areas of Iran.

Studies of drug resistance of Plasmodium falciparum cases from SE Iran were carried out in vivo and in vitro during 1990-91. In a study of 26 chloroquine-resistant infections, using amodiaquine (25 mg kg-1 over 3 days), followed by two-thirds of the standard dose of sulphadoxine-pyrimethamine, six cases were resistant at the RI(3) and RII(3) levels. An additional study testing the standard dose of sulphadoxine-pyrimethamine in 43 chloroquine-resistant cases revealed five (11.6%) resistant at the RI(2) and RII(3) levels. Six of the above resistant infections were contracted in Hormozgan, three in Sistan-Baluchestan, one in Kerman provinces of Iran and one in Pakistan. In five of 22 successful in-vitro tests, carried out on different isolates, the parasites grew to schizonts (> or = 8 nuclei) in the wells containing 1000 pmol sulphadoxine and 12.5 pmol pyrimethamine, indicative of resistance.

Epidemiological aspects of kala-azar in Meshkin-Shahr, Iran: human infection.

During the last decade, kala-azar, which used to be a sporadic disease, became endemic in parts of East-Azerbaijan province in north-west Iran. Many of the 1051 cases observed between 1985 and 1990 were in the district of Meshkin-Shahr (800 cases). The diagnosis was based on symptoms and signs of the disease plus a positive serological test (indirect immunofluorescence assay or direct agglutination test) and, in some cases, the finding of parasites in bone marrow smears. About 90% of the cases were children under 5 years of age. The disease incidence in 1990 was almost ten times that in 1985. New cases are diagnosed throughout the year, but mostly from January till April. Cases were treated with meglumine antimoniate with very satisfactory results; deaths were few among the treated children.

PIP: Sporadic cases of kala-azar have appeared in East-Azerbaijan province, Iran, over the past fifty years. The number of cases began to increase, however, after the mid-1970s and large numbers have been reported from all parts of the country, but mainly in Meshkin-Shahr district. This paper describes the extent of human infection in the district over the period 1984-90, before the implementation of a control program in 1991. 1051 cases were observed in Meshkin-Shahr district hospital over the period 1985-90. 800 of the cases were from Meshkin-Shahr district, with more than 50% of cases being from the city and neighboring larger villages. 90% of cases were children under five years old. 35 cases were diagnosed in 1985 compared to 344 in 1990. New cases were diagnosed throughout the year, but mostly from January to April. Cases were treated with meglumine antimoniate with very satisfactory results; deaths were few among the treated children.

Bacterial infections in suspected cutaneous leishmaniasis lesions.

In Iran, microscopic examination of skin scrapings from 2202 individuals with clinically diagnosed cutaneous leishmaniasis (CL) lesions revealed the presence of amastigotes in 1123 cases (51.0%). Bacteriological examinations of the lesions indicated that 788 individuals (35.7%) were also infected with one or more pathogenic bacteria, including coagulase-positive staphylococci (27.8%), beta-haemolytic streptococci (10.6%), and other opportunist pathogenic bacteria (total, 2.5%). The prevalence of bacterial infections in lesions in which leishmania parasites were detected was 26.5%, while for lesions in which no parasite was found the prevalence of such infections was significantly greater (45%). The results of this study show that bacterial infections should be considered in diagnosing and treating suspected CL lesions, particularly in areas where there is no facility for carrying out bacteriological examinations. Erythromycin can be used to treat the bacterial infections of the purulent sores.

Bacterial infections in suspected cutaneous leshmaniasis lesions

In Iran, microscopic examination of skin scrapings from 2202 individuals with clinically diagnosed cutanesous leishmaniasis (CL) lesions revealed the presence of amastigotes in 1123 cases (51.0 percent)

Bacteriological examinations of the lesions indicated that 788 individuals (35.7 percent) were also infected with one or more pathogenic bacteria, including coagulase-positive staphylococci (27.8 percent), betahaemolytic streptococci (10.6 percent), and other opportunist pathogenic bacteria (total 2.5 percent)

The prevalence of bacterial infections in lesions in which leishmania parasites were detected was 26.5 percent, while for lesions in which no parasite was found the prevalence of such infections was significantly greater (45 percent)

The results of this study show that bacterial infections should be considered in diagnosing and treating suspected CL lesions, particularly in areas where there is no facility for carrying out bacteriological examinations. Erythromycin can be used to treat the bacterial infections of the purulent sores

Status of the response of Plasmodium falciparum to chloroquine and mefloquine in Iran.

Malaria is still the most important parasitic disease in the southeastern part of Iran. The in vivo and in vitro studies of the response of Plasmodium falciparum to chloroquine in Sistan-Baluchestan and Hormozgan provinces (1985-1987) have shown resistance in Iran-Shahr and Bandar Abbas areas. However the parasite was sensitive in Minab area. From 38 chloroquine-resistant P. falciparum (CRPF) cases, found in the studied malarious areas, 13 cases were in Afghan immigrants, 3 in Pakistani and 3 in Iranians who had travelled to Pakistan. Also from 14 CRPF imported cases studied in Teheran, since 1984, four patients were Afghans, 2 Bangalese, one Pakistani and 2 Iranians returned back from India. These findings indicate the great role of migration in introduction and establishment of CRPF in the malarious areas of the southeastern part of Iran. The responses of P. falciparum to mefloquine in the above malarious areas were in general satisfactory. However in few sporadic cases, innate tolerance was observed.

The response of Plasmodium falciparum to chloroquine and mefloquine in Bandar-Abbas and Minab areas, Hormozgan Province, southern Iran.

In-vivo and macro and micro in-vitro assessments of the response of P. falciparum to chloroquine and mefloquine were carried out in the malarious areas of Bandar-Abbas and Minab, Hormozgan Province, southern Iran, during 1986-1987. The susceptibility tests indicated a considerable resistance of P. falciparum to chloroquine in Bandar-Abbas but almost satisfactory sensitivity of the parasite to the drug in Minab. A rather high number of the chloroquine-resistant cases were found among Afghans and Pakistani immigrants and tourists who most probably have introduced and established the resistant strains of the parasite in such malarious areas in Iran. In the micro in-vitro test, P. falciparum was generally sensitive to mefloquine in both Bandar-Abbas and Minab. However, in a few sporadic cases the growth of the parasites occurred in the presence of rather higher doses of the drug, up to 3.2 mumol/l blood, which could be considered to result from innate mefloquine tolerant or resistant strains of P. falciparum in the areas studied.

An endemic focus of visceral leishmaniasis in Meshkin-Shahr, east Azerbaijan province, north-west part of Iran and IFA serological survey of the disease in this area.

The incidence of visceral leishmaniasis has being increased in Iran during the recent decade. Since 1980, more than 200 cases have been diagnosed from East Azerbaijan province, mostly, from Meshkin-Shahr area. It seems, that kala-azar has being endemic in this area for a long time. The majority (86%) of kala-azar cases were found among children up to 4 years. The sex incidence ratio of males/females was 1.27/1. In IFA serological survey, sero-positive rate in females was higher than males. However, geometric mean of leishmanial antibody titers in males was, slightly, more than females. These serological findings indicate that females are exposed to the infection at least as much as males. The cross-sectional IFA serological survey, relatively reflected the kala-azar status among different studied groups with various incidences of the disease in Meshkin-Shahr area. IFAT showed also a good efficiency in the assessment of the treatment in the treated kala-azar patients.

Imported cases of chloroquine-resistant falciparum malaria in Iran.

Six imported cases of chloroquine-resistant Falciparum malaria have been studied since October 1984. In five cases including two Iranian men, returned from India, two Afghan and one Bengalee immigrants came to Iran through Pakistan, recrudescence occurred following treatment with chloroquine. In these five cases resistance of P. falciparum to chloroquine was clinically (by the in vivo test) at R1 level in all patients. The resistance was also confirmed by the macro in vitro susceptibility test which was carried out in four of them. These five chloroquine-resistant cases were treated, one with Sulfadiazine-Pyrimethamine, three with Quinine-Sulfadiazine-Pyrimethamine and one with Sulfadoxine-Pyrimethamine (Fansidar) successfully. In the sixth case who was a Pakistani tourist the parasites showed resistance in the macro in vitro test, but apparently responded to chloroquine treatment in three days. It seems the resistance in this case was also at R1 level as other cases.

Application of an enzyme-linked immunosorbent assay (ELISA) for determination of the human blood index in anopheline mosquitoes collected in Iran.

The microplate method of an ELISA was modified for identification of human blood meals from 5,325 engorged mosquitoes belonging to 12 species of Anopheles captured in 19 provinces of Iran. Four hundred and four (7.5%) specimens reacted with the ELISA anti-human alkaline phosphatase conjugate. The human blood index in nine species of Anopheles varied from 3.6 to 23.7%. The results of this field application of the ELISA indicated that the technique is practical, reproducible and generally a suitable serological test for determination of human blood index of the anopheline mosquitoes.

IFA serological surveys of malaria in north, north-west and south-west parts of Iran.

In the IFA serological surveys of malaria carried out in north, north-west and south-west parts of Iran during 1975-1982 altogether 9,132 subjects were studied for malaria antibodies and parasitaemia. Serological data indicated probable malaria transmission in a consolidation area where the autochthonous cases of malaria were reported a year after this serological study. Asymptomatic parasite carriers of P. malariae were found by IFA and parasite concentration techniques among the professional blood donors and the residents of a village without any recent malaria history. IFA results with P. falciparum antigen reflected the malaria histories in the studied areas of west Azerbaijan better than P. vivax antigen. The serological and parasitological findings in the nomads of Bakhtiary tribes showed that the nomads are more exposed to malaria infection in the winter quarters of Izeh area and they are also more under malaria control programme when they are living in this area. In comparison of IFA results of 438 paired plasma and dried blood samples tested with P. vivax and P. falciparum antigens, there was no significant difference between SPR in plasma and dried blood samples, however in the dried blood samples collected by malaria surveillance agents on filter paper SPR and GMRT were considerably low.