The American Journal of Tropical Medicine and Hygiene - Volume 70, Issue 4, April 2004

Mosquitoes in the Culex (Culex) pipiens complex of species, known as vectors of periodic filariasis and deadly encephalitides, have recently emerged as important vectors of West Nile virus in the United States. Highly conserved morphology but marked differences in potential vectorial capacity require the development of polymerase chain reaction (PCR)-based tests that unambiguously distinguish among the different species. We introduce and describe a series of PCR-based assays that use polymorphisms in the second intron of the acetylcholinesterase-2 (ace-2) locus for the identification of members of the Cx. pipiens complex (Cx. pipiens, Cx. quinquefasciatus, Cx. p. pallens, Cx. australicus), two other species that are commonly mislabeled as Cx. pipiens (Cx. torrentium and Cx. pervigilans), as well as hybrids between Cx. pipiens and Cx. quinquefasciatus.

A high temperature stress of 44.5°C for 10 minutes on the larval stages was found to affect the susceptibility of adult Aedes aegypti mosquitoes to chikungunya virus. At this temperature, the mortality of the mosquito larvae was found to be approximately 95%, whereas a temperature greater than 45°C for 10 minutes was found to be lethal. A temperature tolerant (TT) strain was developed by exposing the larvae to a temperature of 44.5°C for 10 minutes at every generation for five generations. This strain was established to determine whether increase in the susceptibility was due to any selection pressure of higher temperature or to the influence of other intrinsic factors such as expression of immunoresponsive (IR) genes. Other studies on these mosquito strains showed that when maintained at 28 ± 1°C, there was no difference in the larval duration and mortality in the immature stages, but the mean survival of female mosquitoes in the TT strain was 5–6 days longer. Conversely, when mosquitoes were maintained throughout at 37°C the mean survival of the mosquitoes decreased drastically in both strains, but the mean survival of females in the TT strain was 5–6 days longer compared with the unstressed controls. This increases the probability of at least one more blood meal. Fecundity of the TT strain was found to be lower than that of the control mosquitoes. Data suggest that expression of certain IR genes was affected by the heat shock. Some of these genes were up-regulated and down-regulated, which may have affected the susceptibility of mosquitoes to the virus. Although there was some selection in the temperature-tolerant individuals in the TT strain, when stressed by heat they showed expression of IR genes in a pattern similar to that in the normal controls. It appears that an increase in temperature above the average temperature of an area might help increase the proportion of virus-susceptible mosquitoes in the population. Such an increase in temperature in an endemic area would not only enhance the selection of temperature-tolerant individuals in a population having more longevity, but would also affect both intrinsic and extrinsic factors by reducing the extrinsic incubation period and increasing susceptibility of mosquitoes to viruses due to affected expression of IR genes.

Two specific and sensitive polymerase chain reaction (PCR) assays were developed to detect and quantitate Orientia tsutsugamushi, the agent of scrub typhus, using a portion of the 47-kD outer membrane protein antigen/ high temperature requirement A gene as the target. A selected 47-kD protein gene primer pair amplified a 118-basepair fragment from all 26 strains of O. tsutsugamushi evaluated, but it did not produce amplicons when 17 Rickettsia and 18 less-related bacterial nucleic acid extracts were tested. Similar agent specificity for the real-time PCR assay, which used the same primers and a 31-basepair fluorescent probe, was demonstrated. This sensitive and quantitative assay determination of the content of O. tsutsugamushi nucleic acid used a plasmid containing the entire 47-kD gene from the Kato strain as a standard. Enumeration of the copies of O. tsutsugamushi DNA extracted from infected tissues from mice and monkeys following experimental infection with Orientia showed 27-5,552 copies/μL of mouse blood, 14,448-86,012 copies/μL of mouse liver/spleen homogenate, and 3-21 copies/μL of monkey blood.

Between May and October 2002, a cluster of acute febrile illnesses occurred in the subtropical Andean foothills of Peru. Serologic evidence in villages where disease had been documented showed that the prevalence of IgM antibody to Leptospira ranged from 6% to 52%, that of IgM antibody to spotted fever group (SFG) rickettsia ranged from 10% to 19%, and that of IgM antibody to Coxiella burnetii from 1% to 15%. Measurement of IgG antibodies for SFG rickettsiae suggested that this disease was endemic. In contrast, IgG antibodies against C. burnetii were largely absent. In humans, microagglutination tests identified pathogenic variants of Leptospira. The presence of an SFG rickettsial infection was confirmed in four febrile patients following polymerase chain reaction and sequencing of the conserved 17-kD common antigen gene (htrA). Collectively, these analyses indicated that Rickettsia sp., C. burnetii, and Leptospira sp. were circulating in the region during the time of disease outbreak and implicate the involvement of an as yet undetermined SFG rickettsia in northwestern Peru.

The predominant sand fly species collected inside houses in Kfar Adumim, an Israeli village in the Judean Desert that is a focus of cutaneous leishmaniasis, was Phlebotomus papatasi, which was also caught attempting to bite humans. Phlebotomus sergenti, which is rarely seen inside houses, constituted the predominant sand fly species in caves near the village. Leishmania isolates from Ph. sergenti and humans typed as Leishmania tropica. Sand fly and human isolates produced similar small nodular cutaneous lesions in hamsters. Isolates produced excreted factor (EF) of subserotypes A9 or A9B2, characteristic of L. tropica and reacted with L. tropica-specific monoclonal antibodies. Isoenzyme analysis consigned the strains to the L. tropica zymodemes MON-137 and MON-275. Molecular genetic analyses confirmed the strains were L. tropica and intraspecific microheterogeneity was observed. Genomic fingerprinting using a mini-satellite probe separated the L. tropica strains into two clusters that were not entirely congruent with geographic distribution. These results support the heterogeneous nature of L. tropica and incriminate Ph. sergenti as its vector in this Judean Desert focus.

We developed a mathematical model of cutaneous leishmaniasis (CL) transmission predicting CL incidence based on field data of number of positive sand flies, new CL cases, and number of susceptible people. We estimated the following parameters: a CL incubation period of one month, the overall susceptibility (Φ = 0.793), the serologic force of infection (λ m = 0.108 /person/year, SD = 0.014), the clinical force of infection (λ l = 0.114/year), the proportion of infections that result in skin lesions (α = 1.056), and the instantaneous reversal rate of Montenegro skin test-positive (MST+) people to MST− (ρ= 0.124/year, SD = 0.021). We also provide the first field estimate of the transmission efficiency (ε = 0.0045, SD = 0.0009). The model predictions conform well with the observed new cases except for some small departures in the peaks and in some depressions (Dmax = 0.1494, P < 0.2). We discuss possible sources of error of our estimate of ε, and compare our parameter estimates with those obtained in Peru.

This study was designed to examine stool specimens of irritable bowel syndrome (IBS) patients for Blastocystis hominis, a common intestinal parasite. One hundred fifty patients were enrolled, 95 IBS cases and 55 controls. These patients provided a medical history, and underwent physical and laboratory evaluations that included stool microscopy and culture for B. hominis and colonoscopy. The 95 cases (51 males and 44 females) had a mean ± SD age of 37.8 ± 13.2 years. Stool microscopy was positive for B. hominis in 32% (30 of 95) of the cases and 7% (4 of 55) of the controls (P = 0.001). Stool culture was positive in 46% (44 of 95) of the cases and 7% (4 of 55) of the controls (P < 0.001). Stool culture for B. hominis in IBS was more sensitive than microscopy (P < 0.001). Blastocystis hominis was frequently demonstrated in the stool samples of IBS patients; however, its significance in IBS still needs to be investigated. Stool culture has a higher positive yield for B. hominis than stool microscopy.

Myiasis, the invasion of live human tissue by larvae of Diptera, is reported in the nasopharynx and a leg wound in two patients who were hospitalized for more than 72 hours in Mubarak Al-Kabeer Teaching Hospital in Kuwait City, Kuwait. On the fourth and fifth days after a 10-year-old Kuwaiti boy was admitted to the hospital intensive care unit in a bloodied and comatose state following a traffic accident, ‘worms’ that came out of his nostrils were fixed, cleared, and identified as second and third instar of Lucilia sericata (Diptera: Calliphoridae). After 14 days, ‘worms’ were seen in the original dressing of a 35-year-old Iranian man admitted to the Orthopedic Unit of the hospital with multiple lacerations and fractures. The larvae, in various stages of development, were identified as those of Megaselia scalaris (Diptera: Phoridae). Since the presence of larvae in both patients was recorded after a stay of at least 3–4 days in the hospital, by definition, these infestations are considered nosocomial.

We estimated the pre-intervention prevalence of Trypanosoma brucei gambiense (Tbg) trypanosomiasis using the lot quality assurance sampling (LQAS) methods in 14 parishes of Terego County in northern Uganda. A total of 826 participants were included in the survey sample in 1996. The prevalence of laboratory confirmed Tbg trypanosomiasis adjusted for parish population sizes was 2.2% (95% confidence interval =1.1−3.2). This estimate was consistent with the 1.1% period prevalence calculated on the basis of cases identified through passive and active screening in 1996–1999. Ranking of parishes in four categories according to LQAS analysis of the 1996 survey predicted the prevalences observed during the first round of active screening in the population in 1997–1998 (P < 0.0001, by chi-square test). Overall prevalence and ranking of parishes obtained with LQAS were validated by the results of the population screening, suggesting that these survey methods may be useful in the pre-intervention phase of sleeping sickness control programs.

The susceptibility of 20 isolates of Plasmodium vivax on the Thailand-Myanmar border to seven antimalarial drugs was evaluated using the schizont maturation inhibition technique. The geometric mean 50% inhibition concentration (IC50) values were quinine = 308 ng/mL, amodiaquine =14 ng/mL, chloroquine =50 ng/mL, mefloquine = 127 ng/mL, sulfadoxine/pyrimethamine (80:1) = 800/10 ng/mL, pyrimethamine = 8 ng/mL, and artesunate = 0.5 ng/mL. Compared with P. falciparum in this area, P. vivax was more sensitive to chloroquine and artesunate, equally sensitive to quinine, and more resistant to mefloquine.

Placental histopathology was studied in a cohort of 204 women living in an area of low Plasmodium falciparum and P. vivax malaria transmission. Detection of malaria antenatally was active, by weekly peripheral blood smears, and all infections were treated. Significant histopathologic placental malaria changes (increased malaria pigment, cytotrophoblastic prominence, and presence of parasites) were found only in a minority of women who had P. falciparum infections in pregnancy. These changes were significantly more frequent in women with evidence of peripheral blood infection close to delivery and only in these cases were placental inflammatory cells increased. Antenatal P. vivax infection was associated only with the presence of malaria pigment in the placenta. All placental infections diagnosed by blood smear and 32.4% (12 of 37) diagnosed by histopathology were associated with patent peripheral parasitemia. This study indicates that prompt treatment of peripheral parasitemias during pregnancy limits placental pathology. The effect on birth weight reduction may not result from irreversible placental changes but from the acute insult of infection. These findings emphasize the importance of treating malaria in pregnancy promptly with effective antimalarial drugs.

Two previous reviews summarized the New York Hospital experience with 110 cases of malaria from 1968 to 1990. We have extended these studies to include 59 cases of malaria seen from 1991 to 1999 and analyze trends over the past 30 years. Plasmodium falciparum remains the most common species, 38 (64%) of the 59 cases, with the majority of them, 34 (89%) of 38 cases, being acquired in Africa. Of the 59 cases, 22 (37%) were immigrants living in the United States who had visited their countries of origin. Only five (8%) of 59 patients reported using chemoprophylaxis. This represents a marked decrease from the previous reviews. None of the immigrants or their children used chemoprophylaxis. Diagnosis was prompt, and patients responded well to therapy. Complications of malaria were low and no deaths were reported, as was the case in the previous reviews. The low use of chemoprophylaxis, particularly among immigrants, is a major concern.

To determine the response of the small intestinal mucosa to environmental conditions, we studied changes in mucosal architecture and function in a longitudinal cohort study in African adults. Over three consecutive years, 238 adults submitted monthly stool samples for parasitologic and bacteriologic analysis and underwent an annual endoscopic jejunal biopsy for mucosal morphometry. Absorption and permeability assays were performed on the same day as the enteroscopy. Variation in mucosal architecture and function was correlated with environmental factors and stool microbiology. The whole cohort had structural and functional evidence of tropical enteropathy, but structure and function were only weakly correlated. There were marked changes over time, and seasonal variation was observed in villous height (16%), xylose recovery (16%), and permeability (28%). Asymptomatic intestinal infections were common. Enteropathy was more severe in participants with Citrobacter rodentium or hookworm ova in the stool sample taken one month before the investigations were performed.

Hematocrit levels were determined in 36 mothers living at high altitudes (3,750 meters) and their infant cord bloods to determine the effect of maternal anemia on the infant. The arterial oxygen saturation (SaO2) and respiratory rate of the infants were also followed during the first four months of life. There was a negative correlation between maternal hematocrit and infant hematocrit (rs = − 0.57). Nineteen babies born to anemic women (hematocrit < 41%) had a significantly higher mean hematocrit (59.9%) than those born to non-anemic mothers (55.8%; P = 0.003). The SaO2 levels and respiratory rates of infants were not different between infants born to non-anemic and anemic mothers. At high altitudes, infants from mothers with anemia have higher hematocrits than those born to non-anemic mothers.

A fatal case of infection with Angiostrongylus cantonensis is reported in a 14-month-old Jamaican boy. Although infection with Angiostrongylus was not considered initially, sections of multiple worms were observed in the brain and lungs at autopsy and confirmed the infection. This is the first reported fatality due to this infection in the Western Hemisphere, and follows shortly after an outbreak of eosinophilic meningitis among a group of travelers to Jamaica. The source of infection in this case could not be determined.

We report results of the first study to investigate the distribution and diversity of Bartonella in rodents from Thailand. Whole blood from 195 rodents, representing six species, was tested for the presence of Bartonella species using standard culture techniques. Isolates were obtained from 17 (8.7%) of the samples, and 14 of those isolates represented distinct strains, based upon partial sequencing of the citrate synthase (gltA) gene. Phylogenetic analysis of the isolates and other Bartonella species indicated that five unique isolates from Bandicota indica form a cluster that may represent a new Bartonella species. Two additional isolates from B. indica clustered together, and were nearly identical to an isolate from Apodemus draco collected in southern China. Importantly, a number of the isolates from Thailand rodents are closely related to B. grahamii and B. elizabethae, species which have been associated with human illness.

Ethnic differences in the outcome of hepatitis C have been described. Our aim was to investigate ethnic differences in the distribution of genotypes associated with polymorphisms of the tumor necrosis factor-α promoter, interleukin-10 promoter, and transforming growth factor-β1 leader sequence in patients with hepatitis C. Genomic DNA was obtained from 71 Egyptians and 67 Caucasians (hepatitis C and control patients). Amplification of appropriate gene segments was followed by direct sequencing. Infrequently occurring polymorphisms were identified at positions −244 and −77 of the tumor necrosis factor-α promoter and at positions −851 and −657 of the interlukin-10 promoter. The G/A genotype associated with tumor necrosis factor-α promoter positions −376 and −244 was more frequent in Egyptians (P =0.001 and P =0.004, respectively). The −244 G/A genotype occurred only in healthy Egyptians (P =0.024). Thus, ethnic differences in the distribution of genotypes of the tumor necrosis factor-α promoter exist, which may have clinical implications on the outcome of hepatitis C.

Between October 1998 and April 1999, 51 persons belonging to two separate groups developed acute pulmonary histoplasmosis after visiting a cave in Costa Rica. The first group consisted of 61 children and 14 adults from San Jose, Costa Rica; 44 (72%) were diagnosed with acute histoplasmosis. The second group comprised 14 tourists from the United States and Canada; 9 (64%) were diagnosed with histoplasmosis. After a median incubation time of 14 days, the most common symptoms were headache, fever, cough, and myalgias. Risk factors for developing histoplasmosis included crawling (odds ratio [OR] = 17.5, 95% confidence interval [CI] = 2.3–802) and visiting one specific room (OR = 3.4, 95% CI = 1.0–12.3) in the cave. Washing hands (OR = 0.1, 95% CI = 0.01–0.6) after exiting the cave was associated with a decreased risk of developing histoplasmosis. Histoplasma capsulatum was isolated from bat guano collected from inside the cave. Persons who explore caves, whether for recreation or science, should be aware of the risk bat-inhabited caves pose for developing histoplasmosis, especially if they are immunocompromised in any way.

Urinary schistosomiasis remains a major contributor to the disease burden along the southern coast of Kenya. Selective identification of transmission hot spots offers the potential for more effective, highly-focal snail control and human chemotherapy to reduce Schistosoma haematobium transmission. In the present study, a geographic information system was used to integrate demographic, parasitologic, and household location data for an endemic village and neighboring households with the biotic, abiotic, and location data for snail collection/water contact sites. A global spatial statistic was used to detect area-wide trends of clustering for human infection at the household level. Local spatial statistics were then applied to detect specific household clusters of infection, and, as a focal spatial statistic, to evaluate clustering of infection around a putative transmission site. High infection intensities were clustered significantly around a water contact site with high numbers of snails shedding S. haematobium cercariae. When age was considered, clustering was found to be significant at different distances for different age groups.

In the Msambweni area of the Kwale District in Kenya, an area endemic for Schistosoma haematobium, potential intermediate-host snails were systematically surveyed in water bodies associated with human contact that were previously surveyed in the 1980s. Bulinus (africanus) nasutus, which accounted for 67% of the snails collected, was the only snail shedding S. haematobium cercariae. Lanistes purpureus was the second most common snail (25%); lower numbers of Bulinus forskalii and Melanoides tuberculata were also recovered. Infection with non-S. haematobium trematodes was found among all snail species. Rainfall was significantly associated with the temporal distribution of all snail species: high numbers of Bulinus nasutus developed after extensive rainfall, followed, in turn, by increased S. haematobium shedding. Spatial distribution of snails was significantly clustered over a range of up to 1 km, with peak clustering observed at a distance of 400 meters. Water lily (Nymphaea spp.) and several aquatic grass species appeared necessary for local colonization by B. nasutus or L. purpureus.