A conventional multiplex PCR for the detection of four common soil-transmitted nematodes in human feces: development and validation.

Soil-transmitted helminth (STH) infections, mainly caused by Ascaris lumbricoides, Trichuris trichiura, and hookworms, are among the most common intestinal parasites that infect humans. The infections are widely distributed throughout tropical and subtropical countries, including Malaysia, particularly in underprivileged communities. Microscopic and culture techniques have been used as a gold standard for diagnostic techniques. However, these methods yield low sensitivity and specificity, laborious and time-consuming. Therefore, simple, rapid, and accurate alternative methods are needed for the simultaneous detection of STH infections. Although advanced technologies such as real-time multiplex PCR have been established, the use of this technique as a routine diagnostic is limited due to the high cost of the instrument. Therefore, a single-round multiplex conventional PCR assay for rapid detection of four STH species in the fecal sample was developed in this study. To perform the single-round multiplex PCR, each pair of species-specific primers was selected from target genes, including Ancylostoma duodenale (Internal Transcribed Spacer 2; accession No. AJ001594; 156 base pair), Necator americanus (ITS 2; accession No. AJ001599; 225 base pair), Ascaris lumbricoides (Internal Transcribed Spacer 1; accession No. AJ000895; 334 base pair) and Trichuris triciura (partial ITS 1, 5.8s rRNA and partial ITS 2; accession No. AM992981; 518 base pair). The results showed that the newly designed primers could detect the DNA of STH at low concentrations (0.001 ng/ µl) with no cross-amplification with other species. This assay enables the differentiation of single infections as well as mixed infections. It could be used as an alternative and is a convenient method for the detection of STHs, especially for the differentiation of N. americanus and A. duodenale.

A conventional multiplex PCR for the detection of four common soil-transmitted nematodes in human feces: development and validation

@#Soil-transmitted helminth (STH) infections, mainly caused by Ascaris lumbricoides, Trichuris trichiura, and hookworms, are among the most common intestinal parasites that infect humans. The infections are widely distributed throughout tropical and subtropical countries, including Malaysia, particularly in underprivileged communities. Microscopic and culture techniques have been used as a gold standard for diagnostic techniques. However, these methods yield low sensitivity and specificity, laborious and time-consuming. Therefore, simple, rapid, and accurate alternative methods are needed for the simultaneous detection of STH infections. Although advanced technologies such as real-time multiplex PCR have been established, the use of this technique as a routine diagnostic is limited due to the high cost of the instrument. Therefore, a single-round multiplex conventional PCR assay for rapid detection of four STH species in the fecal sample was developed in this study. To perform the single-round multiplex PCR, each pair of species-specific primers was selected from target genes, including Ancylostoma duodenale (Internal Transcribed Spacer 2; accession No. AJ001594; 156 base pair), Necator americanus (ITS 2; accession No. AJ001599; 225 base pair), Ascaris lumbricoides (Internal Transcribed Spacer 1; accession No. AJ000895; 334 base pair) and Trichuris triciura (partial ITS 1, 5.8s rRNA and partial ITS 2; accession No. AM992981; 518 base pair). The results showed that the newly designed primers could detect the DNA of STH at low concentrations (0.001 ng/μl) with no cross-amplification with other species. This assay enables the differentiation of single infections as well as mixed infections. It could be used as an alternative and is a convenient method for the detection of STHs, especially for the differentiation of N. americanus and A. duodenale.

Dengue vector control in Malaysia: Are we moving in the right direction?

Dengue is a major public health problem across more than 123 countries. Vector control has been the hallmark of the dengue control programme in many countries in Southeast Asia since there are no anti-dengue drugs available, and the most recent dengue vaccine is partly efficacious. House-to-house larval surveys, source reduction, larviciding, fogging, ULV which have been carried out since the inception of the dengue control programme in the 1970s are no longer practicable and need to be augmented by more targeted but less ambitious outbreak responses that focus on a few tools that might justify expense of deployment. However, according to recent reports these tools have not really been evaluated for their effectiveness in dengue control. Novel techniques such the release of genetically modified mosquitoes (RIDL) and the use of the bacterium Wolbachia to control the populations of the Ae. aegypti are still under trial. In this review proactive methods to detect epidemics have been suggested. Tools based on adult mosquitoes is an important strategy for dengue vector surveillance and control. The outbreak response may be more efficient when timely vector control measures are implemented after the immediate detection of an infected mosquito.

Distribution of mosquito larvae in various breeding sites in National Zoo Malaysia.

Mosquitoes are principal vectors of major vector-borne diseases. They are widely found throughout urban and rural areas in Malaysia. They are responsible for various vector-borne diseases such as dengue, malaria, filariasis and encephalitis. A total of 158 mosquito larvae specimens were collected from the National Zoo, Malaysia, from 11 types of breeding habitats during the study period from end of May 2007 to July 2007. Aedes albopictus was the predominant species (35.4%), followed by Tripteroides aranoides (26.6%), Lutzia halifaxii (11.4%), Aedes alboscutellatus (10.1%), Aedes caecus (8.9%), Armigeres spp. (4.4%), Malaya genurostris (2.5%) and Culex vishnui (0.6%). It is important to have a mosquito free environment in a public place like the zoo. Routine larval surveillance should be implemented for an effective mosquito control program in order to reduce mosquito population.

Spatial pattern of 2009 dengue distribution in Kuala Lumpur using GIS application.

In the last few years in Malaysia, dengue fever has increased dramatically and has caused huge public health concerns. The present study aimed to establish a spatial distribution of dengue cases in the city of Kuala Lumpur using a combination of Geographic Information System (GIS) and spatial statistical tools. Collation of data from 1,618 dengue cases in 2009 was obtained from Kuala Lumpur City Hall (DBKL). These data were processed and then converted into GIS format. Information on the average monthly rainfall was also used to correlate with the distribution pattern of dengue cases. To asses the spatial distribution of dengue cases, Average Nearest Neighbor (ANN) Analysis was applied together with spatial analysis with the ESRI ArcGIS V9.3 programme. Results indicated that the distribution of dengue cases in Kuala Lumpur for the year 2009 was spatially clustered with R value less than 1 (R = 0.42; z-scores = - 4.47; p < 0.001). Nevertheless, when this pattern was further analyzed according to month by each zone within Kuala Lumpur, two distinct patterns were observed which include a clustered pattern (R value < 1) between April to June and a dispersed pattern (R value > 1) between August and November. In addition, the mean monthly rainfall has not influenced the distribution pattern of the dengue cases. Implementation of control measures is more difficult for dispersed pattern compared to clustered pattern. From this study, it was found that distribution pattern of dengue cases in Kuala Lumpur in 2009 was spatially distributed (dispersed or clustered) rather than cases occurring randomly. It was proven that by using GIS and spatial statistic tools, we can determine the spatial distribution between dengue and population. Utilization of GIS tools is vital in assisting health agencies, epidemiologist, public health officer, town planner and relevant authorities in developing efficient control measures and contingency programmes to effectively combat dengue fever.

Occurrence of Giardia and Cryptosporidium (oo)cysts in the river water of two recreational areas in Selangor, Malaysia.

This study is the first report on the occurrence of Giardia and Cryptosporidium (oo)cysts in recreational rivers water from Malaysia. It was carried out in water samples at two rivers, 'Sungai Congkak' and 'Sungai Batu', located in Selangor State. The occurrence of both Giardia lamblia and Cryptosporidium parvum (oo)cysts was higher in Sungai Congkak (50% or 15/30 and 10% or 3/30 respectively) than Sungai Batu (16% or 5/30 and 3.3% or 1/30 respectively). The mean density of cysts/L was 0.72 in Sungai Congkak and 0.023 in Sungai Batu, and that of oocysts/L was 0.023 in Sungai Congkak and 0.0033 in Sungai Batu, showing that the occurrence of Giardia was higher and more frequent than Cryptosporidium in both rivers. Sungai Congkak also showed higher faecal coliforms count (ranging from 0.48x10³ to 73x10³ CFU/100 mL) than Sungai Batu (0.41x10³ to 16x10³ CFU/100 mL). On the other hand, the Giardia and Cryptosporidium (oo)cysts and faecal coliforms were more concentrated at the downstream station, followed by midstream and upstream stations which might be due to human factors where settlements and recreation areas were located around and between midstream and downstream stations. The (oo)cysts and faecal coliforms also increased during public holidays due to the significantly higher number of visitors (bathers) compared with the week days. All the parameters (physical, faecal coliforms and rainfall) did not show consistent significant correlation (based on r values of Pearson correlation analysis) with both protozoa, therefore these parameters are not suitable as indicator for the presence of Giardia and Cryptosporidium (oo)cysts in both rivers.

Clinical analysis of foreign-born patients with tuberculosis found in Malaysia.

In this retrospective study, we investigated 263 foreign patients who were diagnosed as having tuberculosis at the National Tuberculosis Center (NTBC) from January 2001 to December 2002. The age range was 14-72 years, with a mean of 33.3 +/- 9.95 years. The study subjects were predominantly males (60%) and females comprised 40%, where the greater impact of tuberculosis was observed in the young and active ones (up to 34 years of age), than middle-age (up to 54). A significantly higher percentage of these patients were from the Southeast Asian countries (87%) and particularly occurred in single male (47.5%) and married female (71.4%) patients (p<0.05). We also found that tuberculosis was significantly higher in female (50.5%) and male (64%) with smoking laborers (p<0.05). Fever (70%), cough (90.5%) and BCG vaccination status showed a significantly higher percentage in male patients (p<0.05), whereas lymphadenopathy (22%) was found in a significantly higher percentage in females (p<0.05). Overall, pulmonary disease (94.3%) occurred more commonly in males and the pleura (3.2%) was the most common site of disseminated tuberculosis. By contrast, the lymph node (11.4%) and miliary (4.8%) forms were the more common extrapulmonary tuberculosis in females. More males had higher percentage of treatment completed at > or = 6 (38%) and > or = 9 (13.3%) months in pulmonary tuberculosis, whilst, more females showed higher percentage of treatment completed (8.7%) in extrapulmonary tuberculosis. Surprisingly, more women showed non-compliance to the anti-tubercular therapy than their counterpart in this study.

Polypeptides associated with in vitro cyst formation of Blastocystis hominis.

The objective of this study was to characterize the polypeptides associated with cysts of Blastocystis hominis. This form is believed to be infective and plays a role in parasite resistance to anti-B. hominis drugs currently used for treatment of Blastocystis associated diarrhea. Cysts were induced through in vitro culture of the parasite in complete medium supplemented with bacterial extract with trypticase, metronidazole or doxycycline. SDS-PAGE analysis showed almost similar polypeptide patterns of parasite extracts obtained from in vitro cultured parasites before and after exposure with the three supplements. Polypeptide bands at 76, 58.5, 48, 45, 40, 38, 32, 25 and 22 kDa were constantly seen in all antigenic preparations and no specific cyst-associated polypeptide was present. However, on immunoblot analysis, 3 out of 16 blastocystosis human sera identified a cyst-associated polypeptide at 60 kDa in all parasite extracts prepared from cultures with the three supplements. In addition, there were associated morphological changes detected in these parasites stained with acridine orange and observed under fluorescence microscopy. Metronidazole induced cyst forms (reddish cells) as early as 12 hours post-exposure; more cyst production (with stronger immunoblot bands) occurred after 24 hours exposure. However, cysts rupture with release and destruction of B. hominis daughters cells occurred after 48 hours exposure. Doxycycline induced less cyst-like forms at 24 hours (weaker 60 kDa band) and less destruction of the cysts (60 kDa band still present at 72 hours post exposure). Bacterial extract and trypticase also induced cysts at 12 hours with increasing numbers up to 72 hours exposure (corresponding increase in intensity of 60 kDa band from samples harvested at 12 to 72 hours post exposure) without any sign of deleterious effect on the parasite.