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P Vijayachari

Publications and source records attributed to P Vijayachari.

At least 19 recordsLinked to original sources
PubMed2006

Multilocus sequence typing method for identification and genotypic classification of pathogenic Leptospira species.

BACKGROUND: Leptospira are the parasitic bacterial organisms associated with a broad range of mammalian hosts and are responsible for severe cases of human Leptospirosis. The epidemiology of leptospirosis is complex and dynamic. Multiple serovars have been identified, each adapted to one or more animal hosts. Adaptation is a dynamic process that changes the spatial and temporal distribution of serovars and clinical manifestations in different hosts. Serotyping based on repertoire of surface antigens is an ambiguous and artificial system of classification of leptospiral agents. Molecular typing methods for the identification of pathogenic leptospires up to individual genome species level have been highly sought after since the decipherment of whole genome sequences. Only a few resources exist for microbial genotypic data based on individual techniques such as Multiple Locus Sequence Typing (MLST), but unfortunately no such databases are existent for leptospires. RESULTS: We for the first time report development of a robust MLST method for genotyping of Leptospira. Genotyping based on DNA sequence identity of 4 housekeeping genes and 2 candidate genes was analyzed in a set of 120 strains including 41 reference strains representing different geographical areas and from different sources. Of the six selected genes, adk, icdA and secY were significantly more variable whereas the LipL32 and LipL41 coding genes and the rrs2 gene were moderately variable. The phylogenetic tree clustered the isolates according to the genome-based species. CONCLUSION: The main advantages of MLST over other typing methods for leptospires include reproducibility, robustness, consistency and portability. The genetic relatedness of the leptospires can be better studied by the MLST approach and can be used for molecular epidemiological and evolutionary studies and population genetics.

Animals↗
PubMed2005

Phylogenetic relatedness among leptospiral strains belonging to same serovar recovered from patients with different clinical syndromes.

Leptospirosis is an emerging zoonotic disease with widespread distribution. The disease, caused by a large number of pathogenic serovars of leptospires, varies in severity from mild flu like illness to severe and fatal forms. It has often been observed that the strains of the same serovar are associated with different clinical syndromes. In this study the isolates recovered from patients with mild and severe form of leptospirosis and those isolated from rodents trapped in the same areas were analyzed by using random amplified polymorphic DNA (RAPD) fingerprinting method using the primers PB1, M16, B11 and B12. RAPD fingerprinting patterns of these strains consistently showed five different genetic clusters. Strains belonging to serovar Ratnapura that caused hepato-renal involvement in patients in South India were genetically dissimilar to strains of the same serovar isolated from patients in Andamans who had pulmonary complications. Strains of other serovars causing mild and sever illness could also be discriminated. However, isolates obtained from human patients and rodents in the same geographical areas showed identical fingerprint patterns indicating that strains circulating in different geographical regions, though belonging to same serovar, are unique to each region.

Animals↗
PubMed2005

A clone of Leptospira interrogans sensu stricto is the major cause of leptospirosis in the archipelago of Andaman and Nicobar Islands, India.

AIMS: Andaman and Nicobar Islands in India has a century long history of human leptospirosis. Several isolates have been recovered over the years from different locations. The present study was undertaken to understand the clonal relationship between all these pathogenic leptospires recovered from these islands. METHODS AND RESULTS: Arbitrarily primed polymerase chain reaction (AP-PCR) was employed to genetically characterize 40 isolates recovered during 1995--2001 and their fingerprints were compared with those of 26 reference strains of known genetic and serological affinities. Sequences of PCR-amplified products from representative isolates were compared with those of different strains belonging to seven genospecies. AP-PCR fingerprints revealed that 32 of the 40 isolates were clonal in nature and fingerprints of all the isolates matched with known reference strains of pathogenic Leptospira interrogans sensu stricto. Comparison of sequence data of PCR amplified products of reference strains and isolates also corroborated these findings. CONCLUSIONS: The study revealed that 80% of the isolates recovered from these islands were clonal in nature and all the isolates taken in the study belonged to Leptospira interrogans sensu stricto. SIGNIFICANCE AND IMPACT OF THE STUDY: An extension of the study in animal population would help in understanding the transmission dynamics of this commonly circulating clone in these islands, which in turn might help in effective control of this public health problem.

Base Sequence↗
PubMed2005

Comparison of immunoreactive proteins of commonly circulating serogroups of Leptospira in Andaman Islands, India.

BACKGROUND & OBJECTIVE: Early diagnosis is the key to the treatment of leptospirosis. For development of rapid diagnostic kits, a thorough knowledge about the nature of the proteins expressed by the pathogen during infection is necessary. The present study was undertaken to understand the nature of immunoreactive proteins from commonly circulating serogroups of Leptospira in the endemic Andaman and Nicobar Islands, India. METHODS: Proteins were extracted from six strains of Leptospira representing five different serogroups following four different preparation methods, viz., whole cell lysis by sonication, detergent solubilization, outer and inner membrane isolations, and were subsequently characterized on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblots were made from the sonicated proteins using hyperimmune rabbit antisera, homologous and heterologous patient sera separately. RESULTS: The 67, 65, 45, 43, 35, 32 and 18 kDa major proteins in the whole cell lysate were common among all the five serogroups of Leptospira. The 67, 41, 35, 32, 28 and 22 kDa were the major outer membrane proteins, while 94, 32, 25 and 18 kDa protein were in inner membrane. Immunoblots with hyperimmune rabbit antisera detected 67, 65, 60, 45, 43, 41 and 32 kDa common proteins from the whole cell lysates of all strains while homologous and heterologous patient sera detected 32 kDa as the major immunoreactive protein in all pathogenic serogroups. This protein reacted against specific LipL32 antisera indicating that this protein was LipL32. INTERPRETATION & CONCLUSION: The circulating serogroups of Leptospira have common nature of expression of proteins during human infection. Among several immunoreactive proteins, three (67, 45 and 32 kDa) were recognized as major antigens by both rabbit hyperimmune sera and patients sera while the 32 kDa protein was recognized as the major immunoreactive protein by homologous and heterologous patient sera. These conserved immunoreactive proteins could be utilized in developing indigenous diagnostic tests for leptospirosis.

Animals↗
PubMed2005

Phenotypic & genotypic conservation of ompL1 & lipL41 among leptospiral isolates of Andaman Islands.

BACKGROUND & OBJECTIVES: The leptospiral antigens that are conserved among the diverse pathogenic leptospires have potential importance in the development of new serodiagnostic and immunoprotective strategies. The present study was therefore carried out to find out the phenotypic conservation of the leptospiral proteins OmpL1 and LipL41, and the genetic conservation of ompL1 and lipL41 genes among the leptospiral isolates of Andaman Islands and among the reference strains. METHODS: In one dimensional SDS-PAGE the leptospiral samples prepared from strains of various leptospiral serovars were run and transferred on to nitrocellulose paper and probed with pooled convalescent phase human sera to find out the phenotypic conservation of the protein fragments at 31 and 41 kDa. Further, the proteins were indirectly confirmed as OmpL1 and LipL41 by using specific rabbit hyperimmune sera. Specific primers were utilized to amplify the fragments to study the genetic conservation of ompL1 and lipL41. Further, these two fragments were sequenced and BLAST analysis was done with the whole genome of Leptospira interrogans serovar Lai for comparison. RESULTS: Analysis of individual immunoblots using patient sera showed that the OmpL1 and LipL41 were conserved among all the isolates used in the study. Further, these two proteins were probed with specific rabbit hyperimmune sera of OmpL1 and LipL41 for confirming the fragments and it was found to be conserved among all the isolates. The PCR based amplification further showed that the genes ompL1 and lipL41 were conserved among the leptospiral isolates studied. Sequencing followed by BLAST analysis of these showed 97 per cent similarity with the whole genome sequence and low score values in comparison with other bacterial species. INTERPRETATION & CONCLUSION: The antigenic and genetic conservation of the two proteins, OmpL1 and LipL41, indicated that these could be potential candidates for development of diagnostic test systems for leptospirosis.

Animals↗
PubMed2004

Leptospirosis among schoolchildren of the Andaman & Nicobar Islands, India: low levels of morbidity and mortality among pre-exposed children during an epidemic.

Leptospirosis is an important public health problem in the Andaman Islands. The disease is being increasingly reported among children and adolescents in recent times. An attempt was made to find out the level of exposure to leptospires, to estimate the incidence of infection and to identity the risk factors for acquiring infection among children. A sample of 1544 schoolchildren was selected. Presence of anti-leptospiral antibodies was tested using the microscopic agglutination test (MAT). Students were interviewed for behavioural factors. In total, 341 (221 seronegative and 120 seropositive) students were followed up clinically and serologically during a subsequent outbreak. An overall seropositivity rate of 23.6% (95% CI 21.54-25.81) was observed. Infection rate was 33.5% among seronegatives whereas re-infection rate was 16.7% among seropositives during the outbreak that occurred 1 month after the first sample collection. Morbidity and mortality were found to be higher among seronegative individuals than seropositives. More than 90% of leptospiral infections were found to be subclinical or unnoticed. The high level of exposure among the children results in high infection rates and because they have less previous exposure than adults, they do not have sufficient protection to resist clinical illness during outbreaks.

Adolescent↗
PubMed2004

Percutaneous exposure resulting in laboratory-acquired leptospirosis -- a case report.

A screw-capped glass tube containing a Leptospira culture accidentally broke and the laboratory worker who was handling the tube sustained a cut on his hand. The wound was flooded with the culture. The culture was that of strain MG 347 belonging to serovar Australis recovered from a patient, and it had undergone 52 passages in Ellinghausen McCullough Johnson Harris medium. The laboratory worker developed a headache 21 days after the accident and became febrile the next day. He was hospitalized for 5 days and was treated initially with doxycycline and later with ciprofloxacin. A blood sample collected on the second day of illness, after starting doxycycline therapy, yielded leptospires and the isolate, HZ 651, was identified as serovar Australis. Monoclonal antibody patterns and randomly amplified polymorphic DNA fingerprinting patterns of the isolate and strain MG 347 were identical, thus indicating that HZ 651 and MG 347 were clonal.

Adult↗
PubMed2004

A 22-mer primer enhances discriminatory power of AP-PCR fingerprinting technique in characterization of leptospires.

OBJECTIVES: To evaluate the discriminatory power and usefulness of arbitrarily primed-polymerase chain reaction (AP-PCR) characterization of leptospires with M16 primer. METHODS: AP-PCR fingerprints of 20 reference strains of Leptospira representing 20 different serovars belonging to seven genospecies (Leptospira interrogans, 11; L. noguchii, 2; L. borgpetersenii, 1; L. santarosai, 2; L. biflexa, 2; L. kirschneri, 1; L. weilii, 1) were generated by employing M16 primer. Fingerprints generated with this primer were compared with those generated with two other commonly used primers PB1, and L10. An attempt was also made to type 20 leptospiral isolates with the M16 primer. RESULTS AND CONCLUSION: Fingerprints with M16 primer could not only differentiate between strains of different genospecies, but also between strains of the same genospecies belonging to different serovars. While two commonly used primers (PB1 and L10) failed to discriminate between some of the different serovars belonging to the same genospecies, this primer was able to generate discriminatory fingerprints for all strains tested. All 20 Leptospira isolates, recovered from patients in Andaman Islands, could also be typed by fingerprints generated with the M16 primer. The discriminatory power of M16 primer adds more specificity to the rapidity of this system of characterization and can be used as an excellent tool in epidemiological studies on Leptospira.

Base Sequence↗
PubMed2004

Use of fluorescent amplified fragment length polymorphism for molecular epidemiology of leptospirosis in India.

Nineteen isolates of leptospires recovered from patients during three epidemics that occurred at different places and different times in the Andaman Islands and eight isolates from sporadic cases were characterized using serological and molecular genetic techniques. Group sera and monoclonal antibodies were used for antigenic characterization, whereas fluorescent amplified fragment length polymorphism (FAFLP) was used for genotyping. Of the 27 isolates, 19 were identified as belonging to serogroup Grippotyphosa, 3 belonged to serogroup Australis, 2 belonged to serogroup Icterohaemorrhagiae, and 1 each belonged to serogroups Hebdomadis, Canicola, and Sejroe. Analysis of FAFLP data grouped these 27 isolates into two main clusters of genotypes. One of the clusters, populated by 19 isolates, included 16 outbreak isolates. Seven of these 19 isolates belonged to serovar Ratnapura, 10 belonged to serovar Valbuzzi, and 1 each belonged to serovar Grippotyphosa and serovar Saxkoebing. Of the 27 patients from whom isolates were obtained, 9 had severe illness, and 6 of these 9 patients had pulmonary involvement, 1 had pulmonary and hepatorenal involvement, and the remaining 2 had hepatorenal involvement alone. Two patients out of the nine severe cases died subsequently. The isolates from sporadic cases showed great genetic diversity and were also diverse antigenically. Perhaps the strains belonging to a dominant genotype (the outbreak-associated cluster) possessed epidemic potential and higher virulence with a greater predilection to cause pulmonary complications than strains belonging to other genetic backgrounds.

Base Sequence↗
PubMed2004

Leptospiral proteins expressed during acute & convalescent phases of human leptospirosis.

BACKGROUND & OBJECTIVES: The available serological techniques for the diagnosis of leptospirosis have less sensitivity during the early stage of the disease. Understanding of leptospiral proteins expressed during acute and convalescent phases of leptospirosis, would be help the develop of new serodiagnostic strategies. Therefore, the present study was carried out to identify (i) an antigen that is conserved among the various pathogenic leptospira; (ii) best protein antigen to which immune response can be identified in the acute phase; and (iii) best protein antigen which is present in convalescent sera which can be used for seroepidemiological studies. METHODS: Quantitative immunoblot analysis was performed using acute and convalescent phase human sera along with sera from normal healthy individuals and from patients with typhoid, malaria and hepatitis as the controls. All the samples were analyzed for the leptospiral protein recognition by using IgM and IgG immunoblots. Leptospiral cell fractionation was performed using triton X-114 and lysozyme and further the conservation of leptospiral proteins was also performed. RESULTS: In confirmed cases of leptospirosis, the IgG recognition in acute phase sera was 30.2, 39.5, 27.9, 55.8 and 27.9 per cent for the leptospiral proteins p32, p41/42, p58, p62 and p82 respectively. The IgG has considerably increased to 65.1, 55.8, 46.5, 67.4 and 48.8 per cent against the same proteins during convalescent phase. The IgM recognition was 32.6 , 32.6, 30.2 and 37.2 per cent for acute phase sera and 32.6, 37.2, 44.2 and 41.9 per cent for convalescent phase sera for the leptospiral proteins p14, p25, p32 and p41/42, respectively. Leptospiral proteins like p62 and p82 were recognized among all the control groups with 3.3-15.3 per cent for IgG recognition. INTERPRETATION & CONCLUSION: Leptospiral protein p32 was found to be highly sensitive and specific and could be useful for the development of newer techniques for diagnosis and seroepidemiological studies. Combination of p32 and p41/42 for IgG and p14, p25, p32, p41/42 for IgM would increase the sensitivity of these techniques further.

Antigens, Bacterial↗
PubMed2003

Leptospiral carrier state and seroprevalence among animal population--a cross-sectional sample survey in Andaman and Nicobar Islands.

A study was conducted with the objective of assessing the leptospiral carrier state and seroprevalence among animal population of Andaman and Nicobar Islands. A total of 494 sera samples from different domestic animals and 85 samples from rats (Rattus rattus) were tested by microscopic agglutination test using nine serogroups prevalent in these islands. Antibodies to leptospires were detected in 164 samples giving an overall seroprevalence of (33.11%). The seroprevalence was highest among cows (40.32%). Of 85 rat (Rattus rattus) samples tested for antileptospiral antibodies six (7.1%) were positive. Leptospires were isolated from kidney of two rats and urine of one cow. Isolate from urine of cow was heavily contaminated and was subsequently lost during further subculture. The two isolates were found to be pathogenic, belonging to serogroup Grippotyphosa. The isolates were further characterized by using a set of monoclonal antibodies. The agglutination patterns of isolates were similar to that of ratnapura and valbuzzi, however these did not completely match.

Agglutination Tests↗
PubMed2003

Field application of Lepto lateral flow for rapid diagnosis of leptospirosis.

The Lepto lateral flow assay for leptospirosis was evaluated at a primary health centre in the Andaman Islands, where leptospirosis is endemic. One hundred and seventeen suspected patients were included in the study; acute serum samples were collected from all of them and convalescent samples from 104. The standard criteria for diagnosis of leptospirosis were: (i) isolation of leptospires from blood, (ii) seroconversion in microscopic agglutination test (MAT) with a minimum titre of 100, (iii) a fourfold rise in titre in MAT or (iv) a MAT titre of 400 or more if only a single sample was available. The results of the lateral flow test were compared with these criteria. Lepto lateral flow had sensitivity of 52.9 % (37/70) in the first week of illness and 86 % (49/57) during weeks 2-4. The corresponding specificities were respectively 93.6 % (44/47) and 89.4 % (42/47). The sensitivity was 34.3 % (12/35) on days 2-3 of the illness, 63.3 % (14/22) on days 4-5 and 84.6 % (11/13) at the end of the first week. The test had a positive predictive value of 92.5 % (37/40) during the first week and 90.7 % (49/54) subsequently. Corresponding negative predictive values were respectively 57.1 % (44/77) and 84 % (42/50). Agreement of the results with the standard criteria was low during the first week, but high during weeks 2-4, with a kappa value of 0.7491. The positivity rates of the tests showed a logarithmic relationship with the MAT titres of samples (r(2) = 0.9271). All indices of validity and utility of lateral flow were similar to those of IgM ELISA and Lepto dipstick. The test can be performed at the bedside of the patient, as whole blood can also be used for testing.

Agglutination Tests↗
PubMed2003

Leptospira interrogans serovar Valbuzzi: a cause of severe pulmonary haemorrhages in the Andaman Islands.

Outbreaks of leptospirosis that present with predominant pulmonary signs and symptoms have been occurring in the Andaman Islands since the late 1980s. Before this, pulmonary haemorrhage had not been observed as a common complication of leptospirosis in India. During an outbreak on North Andaman in 1997, four leptospire isolates were obtained from blood of a fatal case and three other patients who recovered. These isolates were characterized using serological and molecular techniques. Cross-agglutination absorption tests and microscopic agglutination tests using mAbs were used for serological characterization. Genetic typing was done using DNA sequencing of PCR products. Serologically, the isolates were closely related to strain Valbuzzi serovar Valbuzzi of serogroup Grippotyphosa. The sequences of PCR products from these isolates were compared with those of 45 strains belonging to seven species. The isolates showed 97.5-100 % sequence similarity to reference strains belonging to Leptospira interrogans, indicating that the isolates belong to L. interrogans. Serogroups Icterohaemorrhagiae and Australis have been incriminated as the cause of pulmonary haemorrhage in China, Korea and Australia. The four isolates characterized in the present study were obtained from patients with similar symptoms. However, they belonged to serovar Valbuzzi of serogroup Grippotyphosa, indicating that serogroups other than Icterohaemorrhagiae and Australis can also cause pulmonary haemorrhage.

Adolescent↗
PubMed2003

Antigenic and genetic relatedness of Leptospira strains isolated from the Andaman Islands in 1929 and 2001.

Leptospirosis is a major public health problem in Andaman Islands. Several strains of Leptospira have been isolated from the Andamans over the years. Leptospires isolated recently from human cases were compared with one of the earliest available isolates from these islands, dating back to 1929, to study their serological and genetic relatedness. Randomly amplified polymorphic DNA (RAPD) fingerprints of the isolates, generated with a primer used previously to differentiate between Leptospira species and serovars, revealed that some of the recent isolates were genetically identical to the 1929 isolate. The antigenic properties of these strains, as revealed by microscopic agglutination tests with group-specific rabbit antisera and mAbs, were also similar. These findings suggest that a Leptospira strain originally isolated in 1929 has possibly persisted in these islands for over 70 years and continues to cause acute leptospirosis in humans.

Agglutination Tests↗
PubMed2002

Evaluation of Lepto Dri Dot as a rapid test for the diagnosis of leptospirosis.

Lepto Dri Dot is a new card agglutination test developed by the Dutch Royal Tropical Institute for the rapid diagnosis of leptospirosis. We evaluated the test in field conditions in The Andaman Islands. Patients suspected of leptospirosis who attended three primary health centres were included in the study. The test results were compared with blood culture or microscopic agglutination tests on paired serum samples; 74 of 124 patients were diagnosed as having leptospirosis based on these criteria. Lepto Dri Dot had a sensitivity of 67.6% (50/74) and a specificity of 66.0% (33/50) during week 1. During weeks 2-4 the values increased to 85.5% (47/55) and 80% (40/50) respectively. An IgM ELISA was also performed on the serum samples for comparison and this was marginally less sensitive, but more specific, during the first week of illness. The positivity rates for the Dri Dot test during days 2-3, 4-5 and 6-7 were 53.1% (17/32), 75.0% (18/24) and 83.3% (15/18), respectively. The corresponding values for ELISA were 28.1% (9/32), 54% (13/24) and 77.8% (14/18). Both Dri Dot and ELISA showed good agreement with the standard diagnostic criteria after the first week of illness (kappa= 0.65 and 0.74, respectively). The overall concordance of the two tests was 89.5 % (kappa = 0.79). The test does not require special storage or sophisticated equipment and can be performed by relatively low skilled personnel.

Agglutination Tests↗
PubMed2001

Evaluation of darkground microscopy as a rapid diagnostic procedure in leptospirosis.

BACKGROUND & OBJECTIVES: Although darkground microscopy (DGM) is the method employed for examining the growth of leptospires in culture, it is not useful for diagnosis when used directly on body fluids. However, it continues to be used as a method for diagnosing leptospirosis in many centres in India. The present study was undertaken to evaluate the diagnostic accuracy of DGM by comparing it with established tests like culture, microscopic agglutination test (MAT), IgM ELISA and Lepto-Dipstick. METHODS: A total of 170 patients clinically suspected to have leptospirosis were included in the study. The gold standard for diagnosis was positive blood culture, or seroconversion, or a four-fold rise in titre in MAT. DGM was done on plasma after centrifugation at 1000 g and 3000 g and on serum. Indices of accuracy including sensitivity, specificity, predictive values and kappa value of agreement with the gold standard diagnostic criteria were calculated. RESULTS: DGM had a sensitivity of 40.2 per cent, specificity of 61.5 per cent, a positive predictive value of 55.2 per cent and a negative predictive value of 46.6 per cent. It had agreement with the gold standard in only 50 per cent cases the kappa value being 0.017141. INTERPRETATION & CONCLUSION: DGM has low indices of accuracy. The results obtained in patients who met the gold standard criteria for diagnosis and those who did not were identical indicating that the test results are not determined by the presence of leptospiral infection in the patient. DGM, therefore, is not recommended as a sole diagnostic procedure for early diagnosis of leptospirosis.

Humans↗
PubMed2001

Evaluation of microscopic agglutination test as a diagnostic tool during acute stage of leptospirosis in high & low endemic areas.

BACKGROUND & OBJECTIVES: Making a diagnosis on the results of a single microscopic agglutination test (MAT) is difficult because of the uncertainties about the cut-off titre. The present study was conducted to determine the significant titre for a single MAT in areas of high and low endemicity for leptospirosis. METHODS: A total of 1944 serum samples were collected from healthy individuals and confirmed patients residing in areas of high and low endemicity. All the sera were screened by MAT using 10 live leptospiral strains as antigens. From the distribution of titres among healthy individuals and in patients, the sensitivity and specificity at different cut-off titres were calculated. Likelihood ratio positive (LR+), likelihood ratio negative (LR-), and LR+/LR- were calculated. Receiver operating characteristics (ROC) curves were plotted for the early and late stages of the disease in both the areas. RESULTS: The ROC plot was totally below the no benefit line during the first week of illness in high endemic area. During the second to fourth weeks it showed better characteristics and the best cut-off titre was 1:200, where the sensitivity was 93.4 per cent and specificity 74.7 per cent LR+ LR- ratio was 41.82 indicating reasonable separation between the positive and negative test results. In the other states the ROC plot was above the no benefit line even during the first week, the best cut-off being 1:50 where the sensitivity was 56.7 per cent and specificity was 90.6 per cent. During the second to fourth weeks the test showed the best characteristics in the low endemicity regions with an ROC curve having the ideal shape. Best cut-off was at 1:100 where the sensitivity was 96.6 per cent and specificity 94.8 per cent LR+ LR- ratio was 523.25 indicating a wide separation between the positive and negative test results. INTERPRETATION & CONCLUSION: MAT does not have any diagnostic value during the first week, particularly in high endemic areas. The best cut-off to be used will be 1:50 in low endemicity areas during the first week, 1:100 during the second to fourth week and 1:200 in high endemicity regions during the second to fourth weeks.

Agglutination Tests↗