Biology & control of Anopheles culicifacies Giles 1901.

Malaria epidemiology is complex due to multiplicity of disease vectors, sibling species complex and variations in bionomical characteristics, vast varied terrain, various ecological determinants. There are six major mosquito vector taxa in India, viz. Anopheles culicifacies, An. fluviatilis, An. stephensi, An. minimus, An. dirus and An. sundaicus. Among these, An. culicifacies is widely distributed and considered the most important vector throughout the plains and forests of India for generating bulk of malaria cases (>60% annually). Major malaria epidemics are caused by An. culicifaices. It is also the vector of tribal malaria except parts of Odisha and Northeastern States of India. An. culicifacies has been the cause of perennial malaria transmission in forests, and over the years penetrated the deforested areas of Northeast. An. culicifacies participates in malaria transmission either alone or along with An. stephensi or An. fluviatilis. The National Vector Borne Disease Control Programme (NVBDCP) spends about 80 per cent malaria control budget annually in the control of An. culicifacies, yet it remains one of the most formidable challenges in India. With recent advances in molecular biology there has been a significant added knowledge in understanding the biology, ecology, genetics and response to interventions, requiring stratification for cost-effective and sustainable malaria control. Research leading to newer interventions that are evidence-based, community oriented and sustainable would be useful in tackling the emerging challenges in malaria control. Current priority areas of research should include in-depth vector biology and control in problem pockets, preparation of malaria-risk maps for focused and selective interventions, monitoring insecticide resistance, cross-border initiative and data sharing, and coordinated control efforts for achieving transmission reduction, and control of drug-resistant malaria. The present review on An. culicifacies provides updated information on vector biology and control outlining thrust areas of research.

Status of DDT and pyrethroid resistance in Indian Aedes albopictus and absence of knockdown resistance (kdr) mutation.

BACKGROUND & OBJECTIVES: Aedes albopictus is one of the vectors for dengue and chikungunya and emergence of pyrethroid resistance in this species could be of a major concern in controlling the vector. This study reports insecticide susceptibility status of Ae. albopictus to DDT and pyrethroids in some Indian populations and status of presence of knockdown resistance (kdr) mutations. METHODS: Three to four day old adult female Ae. albopictus collected from Delhi, Gurgaon (Haryana), Hardwar (Uttarakhand), Guwahati (Assam) and Kottayam (Kerala) were bio-assayed with DDT (4%), permethrin (0.75%) and deltamethrin (0.05%) impregnated papers using WHO standard susceptibility test kit. Mosquitoes were PCRgenotyped for F1534C kdr-mutation in the voltage-gated sodium channel (VGSC) gene. DDT and pyrethroid resistant individuals were sequenced for partial domain II, III and IV of VGSC targeting residues S989, I1011, V1016, F1534 and D1794 where kdr mutations are reported in Ae. aegypti. RESULTS: Adult bioassays revealed varying degree of resistance against DDT among five populations of Ae. albopictus with corrected mortalities ranging between 61 and 92%. Kerala and Delhi populations showed incipient resistance against permethrin and deltamethrin respectively. All other populations were susceptible for both the synthetic pyrethroids. None of the kdr mutations was detected in any of DDT, deltamethrin and permethrin resistant individuals. INTERPRETATION & CONCLUSION: Ae. albopictus has developed resistance against DDT and there is emergence of incipient resistance against pyrethroids in some populations. So far, there is no evidence of presence of knockdown resistance (kdr) mutation in Ae. albopictus.

Phenotypic Characterization and Whole-Genome Analysis of a Novel Bacteriophage HCF1 Infecting Citrobacter amalonaticus and C. freundii.

Citrobacter species often occur in sewage, food, soil, wastewater, and in the intestinal tract of animals and humans. Citrobacter spp. cause urinary tract infections (UTIs) and infantile meningitis in humans. Due to the presence of plasmid-encoded resistance genes, Citrobacter spp. are often resistant to many antibiotics. In this study, Citrobacter virus HCF1, a novel virulent bacteriophage capable of killing Citrobacter amalonaticus and Citrobacter freundii, was isolated from the sewage water. The isolated bacteriophage was characterized with respect to transmission electron microscopy, one-step growth curve, host range, in vitro efficacy, storage stability, and environmental stress tolerance. The one-step growth curve analysis revealed that the latent period of HCF1 was 30 min and the estimated burst size was 121 plaque-forming units (PFU) per bacterial cell. Host range testing indicated that the HCF1 was specific to the Citrobacter genus. In vitro efficacy assay in the effluent of an anaerobic biodigester showed that the HCF1 completely eliminated the host within 4 and 5 h at MOI:100 and MOI:10, respectively, thereby indicating its potential for combating C. amalonaticus infections. The isolated bacteriophage is considerably stable and tolerant to environmental stress. Furthermore, the complete genome of HCF1 was sequenced using Oxford Nanopore sequencing and the data were subjected to detailed bioinformatic analyses. NCBI-BLASTn analysis revealed that the HCF1 genome had a query coverage of 15-21% and a maximum similarity of 77.27-78.49% with 11 bacteriophages of the Drexlerviridae family. Detailed bioinformatic analysis of the genome profile suggests that HCF1 is a novel T1svirus belonging to the Tempevirinae subfamily of the Drexlerviridae family.

Double isotype production by a neoplastic B cell line. II. Allelically excluded production of mu and gamma 1 heavy chains without CH gene rearrangement.

In our accompanying paper, we described a switch variant (BCL1.2.58) that expresses membrane and secreted forms of IgM and IgG1. Both IgM and IgG1 share the same idiotype and use the same VDJ rearrangement. Here, a detailed Southern blot analysis of the entire constant region of the Ig heavy chain (Ig CH) locus of parental (BCL1.B1) and variants (BCL1.B2) DNA showed no detectable rearrangement. Similar analysis of the JH-C mu region led to the conclusion that two heavy chain alleles present in the IgM/IgG1-producing variants carried the same VDJ rearrangement but differed in their 3' flanking regions. One chromosome 12 did not carry any Ig CH genes, whereas, the other chromosome 12 carried one copy of CH genes. In BCL1.B1, however, each of the chromosome 12 alleles carried a full copy of CH genes. Karyotypic analysis confirmed the presence of two translocated t(12;16) chromosomes in both BCL1.2.58 and BCL1.B1 cells, with a break 5' to the VH locus at the distal region (12F2) of chromosome 12, and at the proximal region below the centromere (16B3) of chromosome 16. We conclude that double production of IgM and IgG1 in BCL1.B2 is accomplished by transcription of the corresponding CH genes in germline configuration using a single VDJ on the same chromosome 12.

Comparison of acid and water-soluble chitosan doped fibrous cellulose hemostat wet laid nonwoven web for hemorrhage application.

Fibrous hemostat was produced using wet laying technique comprising of acid and water soluble chitosan with fibrous cellulose. The hemostatic efficiency and their structural properties were evaluated using blood clotting time, whole blood adhesion and analytical characterization techniques. The maximum concentration of chitosan utilised for the production of nonwoven was 1.5 w/v % upon which increased the viscosity of the solution. The absorption of phosphate buffer solution was found to be 1117% for acid soluble chitosan based nonwoven whereas for water soluble chitosan based nonwoven was 997%. Increased red blood cells and platelets were found to be higher in acid soluble chitosan compared to water soluble chitosan based cellulose nonwoven. The blood clotting time for acid soluble chitosan nonwoven was found to be 166 s and water soluble chitosan nonwoven was 170 s. The developed hemostat have potential application in reducing blood loss and also inducing the blood coagulation pathway which was confirmed by the adhesion of platelets on to the surface of nonwoven web.

Characterization of Vibrio cholerae from deep ground water in a cholera endemic area in Central India.

A total of 8 out of 11 deep ground water samples collected from different villages in Central India were found contaminated with Vibrio cholerae non O1, non O139. In a multiplex PCR, isolates were found positive for ompW gene but negative for ctxAB and rfbO1 genes. However, isolates from two places were positive for tcp and zot genes, indicating their intestinal colonization and toxigenic potential. Antibiotic susceptibility studies revealed that all isolates were multidrug resistant. Although, none of the isolates was found PCR positive for the mobile genetic elements, class 1 integrons and SXT constins. The results of this study corroborated that deep ground water can also be an important reservoir of V. cholerae in plane endemic areas, suggesting a continuous monitoring of water samples for timely prevention of the disease.

Complex population history of two Anopheles dirus mosquito species in Southeast Asia suggests the influence of Pleistocene climate change rather than human-mediated effects.

Anopheles dirus and Anopheles baimaii are closely related species which feed on primates, particularly humans, and transmit malaria in the tropical forests of mainland Southeast Asia. Here, we report an in-depth phylogeographic picture based on 269 individuals from 21 populations from mainland Southeast Asia. Analysis of 1537 bp of mtDNA sequence revealed that the population history of A. baimaii is far more complex than previously thought. An old expansion (pre-300 kyr BP) was inferred in northern India/Bangladesh with a wave of south-eastwards expansion arriving at the Thai border (ca 135-173 kyr BP) followed by leptokurtic dispersal very recently (ca 16 kyr BP) into peninsular Thailand. The long and complex population history of these anthropophilic species suggests their expansions are not in response to the relatively recent (ca 40 kyr BP) human expansions in mainland Southeast Asia but, rather, fit well with our understanding of Pleistocene climatic change there.

Porous electrospun starch rich polycaprolactone blend nanofibers for severe hemorrhage.

Starch nanofiber based hemostat was prepared by electrospinning process for biomedical applications. Bead free uniform starch nanofiber along with polycaprolactone polymer was produced using new solvent combination. The swelling of the PCL/starch mat was 240% higher compared to pristine PCL mat. The blood clotting time of the developed PCL/starch mat was 156 s and the contact angle was 30.8°. The results suggest that the developed nanofibers exhibited good hemostatic potential with quick rate of clotting to control blood loss in traumatic injuries.

Semi-nested polymerase chain reaction for detection of toxigenic Vibrio cholerae from environmental water samples.

A rapid and sensitive direct cell semi-nested PCR assay was developed for the detection of viable toxigenic V. cholerae in environmental water samples. The semi-nested PCR assay amplified cholera toxin (ctxA2B) gene present in the toxigenic V. cholerae. The detection sensitivity of direct cell semi-nested PCR was 2 × 10(3) CFU of V. cholerae whereas direct cell single-step PCR could detect 2 × 10(4) CFU of V. cholerae. The performance of the assay was evaluated using environmental water samples after spiking with known number of Vibrio cholerae O1. The spiked water samples were filtered through a 0.22 micrometer membrane and the bacteria retained on filters were enriched in alkaline peptone water and then used directly in the PCR assay. The semi-nested PCR procedure coupled with enrichment could detect less than 1 CFU/ml in ground water and sea water whereas 2 CFU/ml and 20 CFU/ml could be detected in pond water and tap water, respectively. The proposed method is simple, faster than the conventional detection assays and can be used for screening of drinking water or environmental water samples for the presence of toxigenic V. cholerae.

Nucleolus organizers in Mus musculus subspecies and in the RAG mouse cell line.

Silver staining has been used to detect active nucleolus organizer regions (NOR's). By this criterion six mouse chromosomes, numbers 12, 15, 16, 17, 18 and 19, can have an NOR. The number and distribution of chromosomes with NOR's vary among inbred strains of Mus musculus musculus (C57BL/6J, BALB/cJ, C3H/HeJ and C3H/StCpr1BR) and in M. musculus molossinus. In a musculus X molassinus F1 hybrid, nucleolus organizers from each parent are silver stained.--Chromosomes which have NOR's in diploid cells also show them in tetraploid cells and in established cell lines. The BALB/cJ strain shows Ag-staining of NOR's on chromosomes 12, 15, 18 and occasionally 16. In the RAG cell line, which was derived from BALB/c, active NOR's are seen on 12, 15 and 18, even after these chromosomes have undergone structural rearrangements in the cell line. Some correlation exists between the amount of Ag-stain and the size of a secondary construction region, with a large amount of Ag-stain present on a chromosome which has a prominent secondary constriction. There is no correlation between the amount of Ag-stain and the presence or absence of C-band material.

Q- and C-band chromosome markers in inbred strains of Mus musculus.

Differences in the number of chromosomes with secondary constrictions and in the size of the C-band region on certain chromosomes have been observed among the following inbred strains of Mus musculus: C57BL/10J, C57BR/cdJ, DBA/1J, CBA/J, BALB/cJ, and AKR. These differences are useful as indicators of the location of rRNA genes and as normal chromosome markers. The size of each C-band region appears to remain constant over many generations. Only one probable change in the size of a C-band region was found.

Chromosome markers in Mus musculus: strain differences in C-banding.

The mitotic chromosomes of several inbred strains of mice and a series of F(1) hybrids have been analyzed by quinacrine staining and further characterized by the centromeric heterochromatin banding (C-banding). Inbred strains had the same amount of C-banding material on homologous chromosomes but showed variation in the amount on different chromosomes. F(1) hybrids showed characteristics of each parent and it appears that the amount of C-banding on each chromosome is a simple inherited polymorphism. In this study 12 different chromosomes could be distinguished by their C-banding, and these can be used as normal chromosome markers.

Cytological detection of the c-25H deletion involving the albino (c) locus on chromosome 7 in the mouse.

A deletion of the albino (c) locus on mouse chromosome 7 has been demonstrated using Q- and G-banding methods in a mouse heterozygous for the radiation-induced lethal albino allele, c(25H). The deletion, which is thought to be 1-6 cM long, represents about 7.6% of the length of the metaphase chromosome.

Quinacrine fluorescent chromosome analysis of the Snell translocation in the mouse.

The chromosomes involved in the T(2;4)Sn (formerly designated T(5;8) Sn) or Snell translocation in the mouse have been identified as numbers 2 and 4 by analysis of the fluorescent banding patterns of quinacrine mustard-stained chromosomes in primary cultures from heterozygous and homozygous embryos.

Clinical evaluation of a DNA probe assay for the Philadelphia (Ph1) translocation in chronic myelogenous leukemia.

We report the clinical evaluation of an improved DNA probe assay for the characteristic genetic marker of human CML, observed by cytogenetics and designated the Philadelphia chromosome (Ph1). The Ph1 chromosome results from the fusion of c-abl proto-oncogene sequences from chromosome 9 to phl gene sequence on chromosome 22. (The phl gene is often referred to as bcr. However, for clarity we prefer to reserve the designation "bcr" for the region within the phl gene in which translocation breakpoints have been found to occur. We also find it useful to distinguish between two such regions in phl, bcr-210 and bcr-190, named after the 210- and 190-kDa phl/abl fusion proteins resulting from translocations with breakpoints in the respective regions. We refer to the corresponding chromosomal translocations as Ph1(bcr-210) and Ph1(bcr-190).) DNA, extracted from peripheral blood (PB) or bone marrow (BM) and digested with restriction endonuclease BglII, is hybridized with a probe (phl/bcr-3) spanning a breakpoint cluster region within phl. Rearrangements are revealed by the presence of one or two novel junction fragments. Clinical specimens from leukemic patients with active disease were compared by cytogenetic and DNA probe analysis at seven centers in the United States and Europe. The probe assay identified the phl rearrangement in 190 of 191 cases of Ph1-positive CML, as well as in 12 of 27 clinically diagnosed CML specimens lacking a typical Ph1 chromosome. DNA rearrangements also were seen in two of six cases of Ph1-positive ALL. No false positive results were obtained among 93 non-leukemic controls. Mixing experiments showed that the DNA probe assay can detect as few as 1% leukemic cells in a specimen. A preliminary study of CML patients in remission after allogeneic BM transplantation revealed a small fraction of residual Ph1-positive leukemic cells in a significant number of such patients.

Mosquito-borne diseases in Assam, north-east India: current status and key challenges.

Mosquito-borne diseases, including malaria, Japanese encephalitis (JE), lymphatic filariasis and dengue, are major public health concerns in the north-eastern state of Assam, deterring equitable socioeconomic and industrial development. Among these, malaria and JE are the predominant infections and are spread across the state. The incidence of malaria is, however, gradually receding, with a consistent decline in cases over the past few years, although entry and spread of artemisinin-resistant Plasmodium falciparum remains a real threat in the country. JE, formerly endemic in upper Assam, is currently spreading fast across the state, with confirmed cases and a high case-fatality rate affecting all ages. Lymphatic filariasisis is prevalent but its distribution is confined to a few districts and disease transmission is steadily declining. Dengue has recently invaded the state, with a large concentration of cases in Guwahati city that are spreading to suburban areas. Control of these diseases requires robust disease surveillance and integrated vector management on a sustained basis, ensuring universal coverage of evidence-based key interventions based on sound epidemiological data. This paper aims to present a comprehensive review of the status of vector-borne diseases in Assam and to address the key challenges.

Dengue, an emerging arboviral infection in Assam, northeast India.

Dengue is emerging as major public health concern in northeast India and spreading with increased morbidity. Most cases were recorded in Guwahati metropolitan city of the state of Assam during post-monsoon months (September- December). These comprised all age groups of both sexes with significantly higher incidence of cases in adult males aged 26- 60 years.

Left ventricular diastolic function in end-stage renal disease and the impact of hemodialysis.

Twenty-one patients (17 men and 4 women, aged 20 to 40 years) with end-stage renal disease (creatinine clearance persistently < 5 ml/min for > 3 months) were evaluated for left ventricular (LV) diastolic function by Doppler echocardiography before and after hemodialysis. Fifteen patients were on maintenance hemodialysis (group A) and 6 were studied before and after their first hemodialysis (group B). The following indexes of LV diastolic function were studied: (1) isovolumic relaxation time; and (2) Doppler indexes from mitral inflow signal--peak early velocity, peak late velocity (atrial), deceleration of early filling phase, and deceleration time of early filling phase. LV systolic function in groups A and B (LV ejection fraction 68 +/- 6 and 77 +/- 5%, fractional shortening 0.39 +/- 0.06 and 0.46 +/- 0.05%) was normal and did not change after hemodialysis. Group A had a prolonged isovolumic relaxation time of 80 +/- 22 ms, which decreased to 57 +/- 14 ms (p < 0.005). Deceleration time decreased from 248 +/- 58 to 184 +/- 38 ms (p < 0.00005) and the deceleration slope increased from 4.3 +/- 1.8 to 5.1 +/- 1.6 m/s2 (p < 0.005) after hemodialysis. In group B, isovolumic relaxation time decreased from 87 +/- 21 to 73 +/- 15 ms (p < 0.05), deceleration time decreased from 256 +/- 43 to 185 +/- 34 ms (p < 0.05), and deceleration slope increased from 3.5 +/- 0.8 to 4.2 +/- 1.1 m/s2 (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Allelic variation in the cg2 gene does not correlate with chloroquine resistance among Indian Plasmodium falciparum isolates.

The cg2 gene of Plasmodium falciparum has been proposed to be associated with chloroquine resistance. Here we describe PCR amplification and sequencing of all the four repeat regions (kappa (kappa), gamma (gamma), psi (psi) and omega (omega)) of this gene, from Indian isolates. There were variant forms for each of these repeat regions (two for kappa and gamma, and three for psi and omega) among the 123 Indian isolates of P. falciparum. Among these isolates certain forms of psi and omega repeats were uniquely present while some of the reported forms of the kappa and omega repeats were absent. The pattern of combination of all four repeat regions of cg2 gene (genotype) was analysed from 52 isolates. A total of 11 different genotypes were observed among these cases, of which 10 were unique to Indian isolates. Certain genotypes were more common than others. The nucleotide sequencing of all the four repeat regions revealed that Indian isolates have some unique repeating units within the gamma and omega domains. Altogether, the PCR and sequencing results showed that there was an unrelatedness between cg2 repeats and chloroquine resistance.

Direct karyotyping of unstimulated newborn blood: a rapid diagnostic method for the clinical management of critically ill newborns.

Using spontaneously dividing nucleated erythrocytes present in newborn cord and peripheral blood, we performed direct karyotype analysis on a cohort of 162 infants suspected of chromosome abnormalities. A cytogenetic diagnosis was obtained in 149 cases (91.9%). In all cases conventional phytohaemagglutinin- (PHA)-stimulated cultures were used for comparison. Concordance between direct and stimulated karyotypes was seen in all but 5 cases. In these 5 cases, abnormalities were seen in the direct harvest while PHA-stimulated cultures showed normal results. Skin fibroblasts from 2 of these cases, available for follow-up, showed the abnormalities in a mosaic state. Our experience confirms that direct karyotyping of fetal and newborn blood is feasible, fast, and efficient and can provide accurate diagnosis of major chromosome abnormalities within 18-24 hours after obtaining the blood.