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Fulltext Detection of Naegleria species in environmental samples from Peninsular Malaysia

Ithoi I, Ahmad AF, Nissapatorn V, Lau YL, Mahmud R, Mak JW

PLoS One, 2011;6(9):e24327.
PMID: 21915311 DOI: 10.1371/journal.pone.0024327

BACKGROUND: In Malaysia, researchers and medical practitioners are unfamiliar with Naegleria infections. Thus little is known about the existence of pathogenic Naegleria fowleri, and the resultant primary amoebic meningoencephalitis (PAM) is seldom included in the differential diagnosis of central nervous system infections. This study was conducted to detect the presence of Naegleria species in various environmental samples.
METHODS/FINDINGS: A total of 41 Naegleria-like isolates were isolated from water and dust samples. All these isolates were subjected to PCR using two primer sets designed from the ITS1-ITS2 regions. The N. fowleri species-specific primer set failed to produce the expected amplicon. The Naegleria genus-specific primers produced amplicons of 408 bp (35), 450 bp (2), 457 bp (2) or 381 bp (2) from all 41 isolates isolated from aquatic (33) and dust (8) samples. Analysis of the sequences from 10 representative isolates revealed that amplicons with fragments 408, 450 and 457 bp showed homology with non-pathogenic Naegleria species, and 381 bp showed homology with Vahlkampfia species. These results concurred with the morphological observation that all 39 isolates which exhibited flagella were Naegleria, while 2 isolates (AC7, JN034055 and AC8, JN034056) that did not exhibit flagella were Vahlkampfia species.
CONCLUSION: To date, pathogenic species of N. fowleri have not been isolated from Malaysia. All 39 isolates that produced amplicons (408, 450 and 457 bp) from the genus-specific primers were identified as being similar to nonpathogenic Naegleria. Amplicon 408 bp from 5 representative isolates showed 100% and 99.7% identity to Naegleria philippinensis isolate RJTM (AM167890) and is thus believed to be the most common species in our environment. Amplicons 450 bp and 457 bp were respectively believed to be from 2 new species of Naegleria, since representative isolates showed lower homology and had a longer base pair length when compared to the reference species in the Genbank, Naegleria schusteri (AJ566626) and Naegleria laresi (AJ566630), respectively.
Fulltext The Authentication and Grading of Edible Bird's Nest by Metabolite, Nutritional, and Mineral Profiling

Mohamad Ibrahim R, Mohamad Nasir NN, Abu Bakar MZ, Mahmud R, Ab Razak NA

Foods, 2021 Jul 07;10(7).
PMID: 34359444 DOI: 10.3390/foods10071574

Edible bird's nest (EBN) produced by Aerodramus fuciphagus has a high demand for nutritional and medicinal application throughout the world. The present study was to evaluate the authentication of a man-made house EBN, which are half cup and stripe-shaped by FTIR. Next, both samples were compared according to their metabolite, nutritional, and mineral composition. The results indicated that the FTIR spectra of both EBN samples were identical and similar to the reference, suggesting the authenticity of the EBN used. The metabolites that contribute to the possible medicinal properties of EBN were found by using GC-MS. The results of the proximate analysis, followed by the standard AOAC method, inferred that both EBN shapes to be rich in crude protein and carbohydrate contents. However, the proximate composition between the half cup and stripe-shaped EBN showed significant differences. Major mineral elements detected were calcium and sodium, and magnesium contents were significantly different between both EBN. Additionally, the half cup and stripe-shaped EBN had a low level of heavy metal content than the maximum regulatory limit as set by the Malaysian Food Act 1983. This study concludes that the nutritional composition varied between the samples and thus suggests that nutrient content should be considered as criteria for the grading requirement of commercialized EBN.
Fulltext Effects of Counselling on Quality of Life among Cancer Patients in Malaysia: A Randomized Controlled Trial

Periasamy U, Mohd-Sidik S, Akhtari-Zavare M, Rampal L, Ismail SIF, Mahmud R

Iran J Public Health, 2020 Oct;49(10):1902-1911.
PMID: 33346212 DOI: 10.18502/ijph.v49i10.4693

Background: We aimed to assess whether "Managing Patients on Chemotherapy" book is effective to improve quality of life (QOL) of cancer patient via counselling by pharmacist.
Methods: A randomized control trial study was run among 2120 cancer patients in public hospitals in Peninsular Malaysia, from Apr 2016-Jan 2018. The treatment group received counselling regarding chemotherapy by using developed module. The data were collected at three time-points: baseline, 1st, 2nd and 3rd follow-ups after counseling by Validated Malay version of the WHOQOL-BREF of questionnaire. Data analyses were done using χ2 and two-way repeated measure ANOVA.
Results: The treatment group improved significantly as compared to control group in physical health, psychological health, social relationship, environment and overall QOL (P<0.00).
Conclusion: The "Managing Patients on Chemotherapy" book along with repetitive counselling by pharmacists is a useful intervention for improving QOL of cancer patients undergoing treatment.
Sonographic and clinical effects of manual physical therapy for plantar fasciitis: randomized prospective controlled trial

Khammas ASA, Mahmud R, Hassan HA, Ibrahim I, Mohammed SS

J Ultrasound, 2024 Sep;27(3):487-500.
PMID: 37917407 DOI: 10.1007/s40477-023-00833-0

OBJECTIVE: This study aimed to evaluate the efficacy of manual physiotherapy on clinical outcomes, morphology of plantar fascia (PF), thicknesses of calcaneal fat pad (CFP) and Kager's fat pad (KFP) with ultrasound imaging in plantar fasciitis (PFS) patients. Also, to evaluate the PF thickness, pain and foot functional outcomes among PFS phases.
METHODS: A randomized controlled trial was conducted on 122 subjects divided into three groups: group A (40 patients with PFS) underwent manual physiotherapy, group B (42 patients with PFS) without any intervention and group C (40 healthy subjects) were matched by age, gender and BMI with each patient in group A and B. The following outcomes were evaluated at baseline and one-month of follow-ups: morphology of PF and thicknesses of CFP and KFP, pain, foot functional limitation.
RESULTS: PF thickness was significantly thickened in group A and B compared to group C (P
Fulltext Laboratory Evaluation of a Rapid IgG4 Antibody Test (BLF Rapid™) for Bancroftian Filariasis

Noordin R, Yunus MH, Robinson K, Won KY, Babu S, Fischer PU, et al.

Am J Trop Med Hyg, 2018 12;99(6):1587-1590.
PMID: 30350768 DOI: 10.4269/ajtmh.18-0566

At the end phase of the Global Programme to Eliminate Lymphatic Filariasis, antibody testing may have a role in decision-making for bancroftian filariasis-endemic areas. This study evaluated the diagnostic performance of BLF Rapid™, a prototype immunochromatographic IgG4-based test using BmSXP recombinant protein, for detection of bancroftian filariasis. The test was evaluated using 258 serum samples, comprising 96 samples tested at Universiti Sains Malaysia (in-house) and 162 samples tested independently at three international laboratories in the USA and India, and two laboratories in Malaysia. The independent testing involved 99 samples from Wuchereria bancrofti microfilaria or antigen positive individuals and 63 samples from people who were healthy or had other infections. The in-house evaluation showed 100% diagnostic sensitivity and specificity. The independent evaluations showed a diagnostic sensitivity of 84-100% and 100% specificity (excluding non-lymphatic filarial infections). BLF Rapid has potential as a surveillance diagnostic tool to make "Transmission Assessment Survey"-stopping decisions and conduct post-elimination surveillance.
Fulltext Molecular Diagnosis of Microsporidia among Immunocompromised Patients in Kuala Lumpur, Malaysia

Hassan NA, Lim YAL, Mahmud R, Mohd-Shaharuddin N, Wan Sulaiman WY, Ngui R

Am J Trop Med Hyg, 2018 Dec;99(6):1562-1566.
PMID: 30382015 DOI: 10.4269/ajtmh.17-0901

Microsporidia are obligate intracellular parasitic fungi causing chronic diarrhea, particularly among immunocompromised patients. The current method used for diagnosis is based on conventional microscopy, which does not differentiate parasites at the species level. The present study was carried out to identify microsporidian species in immunocompromised patients. From March 2016 to March 2017, a total of 289 archived stool samples were examined microscopically for microsporidian spores using Gram-chromotrope Kinyoun (GCK) stain. Positive stool samples by microscopy were subjected to polymerase chain reaction and sequencing for species identification. Based on microscopy examination, the overall prevalence of microsporidian infections was 32.2% (93/289; 95% CI = 27.1-37.8). Of these stool samples, 45 samples were successfully amplified and confirmed as Enterocytozoon bieneusi. No Encephalitozoon intestinalis was detected. Accurate identification of species might help clinicians to decide appropriate management strategies as dissemination risks and treatment response vary for different species, hence improving the management of microsporidian infections.
Fulltext Seroprevalence and sources of toxoplasmosis among Orang Asli (indigenous) communities in Peninsular Malaysia

Ngui R, Lim YA, Amir NF, Nissapatorn V, Mahmud R

Am J Trop Med Hyg, 2011 Oct;85(4):660-6.
PMID: 21976569 DOI: 10.4269/ajtmh.2011.11-0058

This study aims to evaluate the current seroprevalence of toxoplasmosis among indigenous communities in Peninsular Malaysia and relate its association with epidemiological data. Overall seroprevalence of Toxoplasma gondii was 37.0% with 31.0% immunoglobulin (Ig) G, 1.8% IgM, and 4.2% seropositivity for both anti-Toxoplasma antibodies. Multivariate analysis showed that age above 12 years (odds ratio [OR] = 2.70, 95% confidence interval [CI] = 1.75-4.04, P < 0.001), using untreated river and mountain water supplies (OR = 1.50, 95% CI = 1.01-2.40, P = 0.050), and close proximity with cats (OR = 1.40, 95% CI = 1.10-1.76, P = 0.010) were factors associated with toxoplasmosis. Given the high seroprevalence of toxoplasmosis among these communities who live in poor socioeconomic conditions, a comprehensive health surveillance program and screening should be initiated among women of childbearing age and pregnant women during the antenatal period for early diagnosis and treatment. The role of domestic cats and environmental contamination with oocyst in soil and water has to be highlighted and addressed in future prevention strategies for these communities.
Fulltext Comparison of three molecular methods for the detection and speciation of five human Plasmodium species

Lau YL, Lai MY, Anthony CN, Chang PY, Palaeya V, Fong MY, et al.

Am J Trop Med Hyg, 2015 Jan;92(1):28-33.
PMID: 25385862 DOI: 10.4269/ajtmh.14-0309

In this study, three molecular assays (real-time multiplex polymerase chain reaction [PCR], merozoite surface antigen gene [MSP]-multiplex PCR, and the PlasmoNex Multiplex PCR Kit) have been developed for diagnosis of Plasmodium species. In total, 52 microscopy-positive and 20 malaria-negative samples were used in this study. We found that real-time multiplex PCR was the most sensitive for detecting P. falciparum and P. knowlesi. The MSP-multiplex PCR assay and the PlasmoNex Multiplex PCR Kit were equally sensitive for diagnosing P. knowlesi infection, whereas the PlasmoNex Multiplex PCR Kit and real-time multiplex PCR showed similar sensitivity for detecting P. vivax. The three molecular assays displayed 100% specificity for detecting malaria samples. We observed no significant differences between MSP-multiplex PCR and the PlasmoNex multiplex PCR kit (McNemar's test: P = 0.1489). However, significant differences were observed comparing real-time multiplex PCR with the PlasmoNex Multiplex PCR Kit (McNemar's test: P = 0.0044) or real-time multiplex PCR with MSP-multiplex PCR (McNemar's test: P = 0.0012).
Fulltext Sarcocystis nesbitti infection in human skeletal muscle: possible transmission from snakes

Lau YL, Chang PY, Tan CT, Fong MY, Mahmud R, Wong KT

Am J Trop Med Hyg, 2014 Feb;90(2):361-4.
PMID: 24420776 DOI: 10.4269/ajtmh.12-0678

Sarcocystis nesbitti is an intracellular protozoan parasite found as sarcocysts within muscle fibers of intermediate hosts (monkey and baboon). The definitive host is suspected to be the snake. We report two cases from a larger cohort of 89 patients who had fever, headache, and generalized myalgia after a trip to Pangkor Island, Malaysia. Sarcocysts were detected in skeletal muscle biopsy specimens by light and electron microscopy from these two patients. DNA sequencing based on the 18S ribosomal DNA region identified the Sarcocystis species as S. nesbitti. We also identified S. nesbitti sequences in the stools of a snake (Naja naja). Phylogenetic analysis showed that these sequences form a cluster with most of the other known Sarcocystis species for which the snake is a definitive host. We believe these two patients were likely to have symptomatic acute muscular sarcocystosis after S. nesbitti infection that may have originated from snakes.
Fulltext Zoonotic Ancylostoma ceylanicum infection detected by endoscopy

Ngui R, Lim YA, Ismail WH, Lim KN, Mahmud R

Am J Trop Med Hyg, 2014 Jul;91(1):86-8.
PMID: 24891471 DOI: 10.4269/ajtmh.13-0756

We report a case of Ancylostoma ceylanicum infection detected by endoscopy. It was diagnosed and confirmed using polymerase chain reaction (PCR) and DNA sequencing. The patient is a 58-year-old Malaysian woman who lives in a rural area, where uncontrolled populations of stray and semidomesticated dogs live in close proximity with humans.
Fulltext High seroprevalence of echinococossis, schistosomiasis and toxoplasmosis among the populations in Babati and Monduli districts, Tanzania

Khan MB, Sonaimuthu P, Lau YL, Al-Mekhlafi HM, Mahmud R, Kavana N, et al.

Parasit Vectors, 2014;7:505.
PMID: 25388913 DOI: 10.1186/s13071-014-0505-7

The neglected tropical diseases, echinococcosis, schistosomiasis and toxoplasmosis are all globally widespread zoonotic diseases with potentially harmful consequences. There is very limited data available on the prevalence of these infections, except for schistosmiasis, in underdeveloped countries. This study aimed to determine the seroprevalence of Echinococcus multilocularis, Schistosoma mansoni, and Toxoplasma gondii antibodies in populations from the Monduli and Babati districts in Tanzania.
Fulltext Developing and evaluating health education learning package (HELP) to control soil-transmitted helminth infections among Orang Asli children in Malaysia

Al-Delaimy AK, Al-Mekhlafi HM, Lim YA, Nasr NA, Sady H, Atroosh WM, et al.

Parasit Vectors, 2014;7:416.
PMID: 25179100 DOI: 10.1186/1756-3305-7-416

This study was carried out to develop a health education learning package (HELP) about soil-transmitted helminth (STH) infections, and to evaluate what impact such a package could have in terms of reducing the incidence and intensity of STH infections among Orang Asli schoolchildren in Pahang, Malaysia.
Fulltext High proportion of knowlesi malaria in recent malaria cases in Malaysia

Yusof R, Lau YL, Mahmud R, Fong MY, Jelip J, Ngian HU, et al.

Malar J, 2014;13:168.
PMID: 24886266 DOI: 10.1186/1475-2875-13-168

Plasmodium knowlesi is a simian parasite that has been recognized as the fifth species causing human malaria. Naturally-acquired P. knowlesi infection is widespread among human populations in Southeast Asia. The aim of this epidemiological study was to determine the incidence and distribution of malaria parasites, with a particular focus on human P. knowlesi infection in Malaysia.
Mapping and modelling the geographical distribution of soil-transmitted helminthiases in Peninsular Malaysia: implications for control approaches

Ngui R, Shafie A, Chua KH, Mistam MS, Al-Mekhlafi HM, Sulaiman WW, et al.

Geospat Health, 2014 May;8(2):365-76.
PMID: 24893014

Soil-transmitted helminth (STH) infections in Malaysia are still highly prevalent, especially in rural and remote communities. Complete estimations of the total disease burden in the country has not been performed, since available data are not easily accessible in the public domain. The current study utilised geographical information system (GIS) to collate and map the distribution of STH infections from available empirical survey data in Peninsular Malaysia, highlighting areas where information is lacking. The assembled database, comprising surveys conducted between 1970 and 2012 in 99 different locations, represents one of the most comprehensive compilations of STH infections in the country. It was found that the geographical distribution of STH varies considerably with no clear pattern across the surveyed locations. Our attempt to generate predictive risk maps of STH infections on the basis of ecological limits such as climate and other environmental factors shows that the prevalence of Ascaris lumbricoides is low along the western coast and the southern part of the country, whilst the prevalence is high in the central plains and in the North. In the present study, we demonstrate that GIS can play an important role in providing data for the implementation of sustainable and effective STH control programmes to policy-makers and authorities in charge.
Fulltext A randomised controlled trial on hypolipidemic effects of Nigella Sativa seeds powder in menopausal women

Ibrahim RM, Hamdan NS, Mahmud R, Imam MU, Saini SM, Rashid SN, et al.

J Transl Med, 2014;12:82.
PMID: 24685020 DOI: 10.1186/1479-5876-12-82

The risk of cardiovascular diseases (CVD) is increased tremendously among menopausal women, and there is an increasing demand for alternative therapies for managing factors like dyslipidemia that contribute to CVD development.
Fulltext Malaysian child infected with Plasmodium vivax via blood transfusion: a case report

Anthony CN, Lau YL, Sum JS, Fong MY, Ariffin H, Zaw WL, et al.

Malar J, 2013;12:308.
PMID: 24007496 DOI: 10.1186/1475-2875-12-308

Malaria may be a serious complication of blood transfusion due to the asymptomatic persistence of parasites in some donors. This case report highlights the transfusion-transmitted malaria of Plasmodium vivax in a child diagnosed with germ cell tumour. This child had received blood transfusion from three donors and a week later started developing malaria like symptoms. Nested PCR and sequencing confirmed that one of the three donors was infected with P. vivax and this was transmitted to the 12-year-old child. To the best of the authors' knowledge, this is the first reported transfusion-transmitted malaria case in Malaysia.
Multilocus genotyping of Giardia duodenalis in Malaysia

Huey CS, Mahdy MA, Al-Mekhlafi HM, Nasr NA, Lim YA, Mahmud R, et al.

Infect Genet Evol, 2013 Jul;17:269-76.
PMID: 23624189 DOI: 10.1016/j.meegid.2013.04.013

Giardia duodenalis is considered the most common intestinal parasite in humans worldwide. In Malaysia, many studies have been conducted on the epidemiology of giardiasis. However, there is a scarcity of information on the genetic diversity and the dynamics of transmission of G. duodenalis. The present study was conducted to identify G. duodenalis assemblages and sub-assemblages based on multilocus analysis of the glutamate dehydrogenase (gdh), beta-giardin (bg) and triose phosphate isomerase (tpi) genes. Faecal specimens were collected from 484 Orang Asli children with a mean age of 7 years and examined using light microscopy. Specimens positive for Giardia were subjected to PCR analysis of the three genes and subsequent sequencing in both directions. Sequences were edited and analysed by phylogenetic analysis. G. duodenalis was detected in 17% (84 of 484) of the examined specimens. Among them, 71 were successfully sequenced using at least one locus. Genotyping results showed that 30 (42%) of the isolates belonged to assemblage A, 32 (45%) belonged to assemblage B, while discordant genotype results were observed in 9 specimens. Mixed infections were detected in 43 specimens using a tpi-based assemblage specific protocol. At the sub-assemblages level, isolates belonged to assemblage A were AII. High nucleotide variation found in isolates of assemblage B made subtyping difficult to achieve. The finding of assemblage B and the anthroponotic genotype AII implicates human-to-human transmission as the most possible mode of transmission among Malaysian aborigines. The high polymorphism found in isolates of assemblage B warrants a more defining tool to discriminate assemblage B at the sub-assemblage level.
Fulltext Hyperparasitaemic human Plasmodium knowlesi infection with atypical morphology in peninsular Malaysia

Lee WC, Chin PW, Lau YL, Chin LC, Fong MY, Yap CJ, et al.

Malar J, 2013;12:88.
PMID: 23496970 DOI: 10.1186/1475-2875-12-88

Plasmodium knowlesi is a potentially life-threatening zoonotic malaria parasite due to its relatively short erythrocytic cycle. Microscopic identification of P. knowlesi is difficult, with "compacted parasite cytoplasm" being one of the important identifying keys. This report is about a case of hyperparasitaemic human P. knowlesi infection (27% parasitaemia) with atypical amoeboid morphology. A peninsular Malaysian was admitted to the hospital with malaria. He suffered anaemia and acute kidney function impairment. Microscopic examination, assisted by nested PCR and sequencing confirmed as P. knowlesi infection. With anti-malarial treatment and several medical interventions, patient survived and recovered. One-month medical follow-up was performed after recovery and no recrudescence was noted. This case report highlights the extreme hyperparasitaemic setting, the atypical morphology of P. knowlesi in the patient's erythrocytes, as well as the medical interventions involved in this successfully treated case.
Fulltext A simple isocratic HPLC method for the simultaneous determination of sinensetin, eupatorin, and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone in Orthosiphon stamineus extracts

Yam MF, Mohamed EA, Ang LF, Pei L, Darwis Y, Mahmud R, et al.

J Acupunct Meridian Stud, 2012 Aug;5(4):176-82.
PMID: 22898066 DOI: 10.1016/j.jams.2012.05.005

Orthosiphon stamineus extracts contain three flavonoids (3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin) as bioactive substances. Previous reported high performance liquid chromatography- ultraviolet (HPLC-UV) methods for the determination of these flavonoids have several disadvantages, including unsatisfactory separation times and not being well validated according to International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) standard guidelines. A rapid, specific reversed-phase HPLC method with isocratic elution of acetonitrile: isopropyl alcohol: 20mM phosphate buffer (NaH(2)PO(4)) (30:15:55, v/v) (pH 3.5) at a flow-rate of 1ml/minute, a column temperature of 25°C, and ultraviolet (UV) detection at 340 nm was developed. The method was validated and applied for quantification of different types of O stamineus extracts and fractions. The method allowed simultaneous determination of 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin in the concentration range of 0.03052-250 μg/ml. The limits of detection and quantification, respectively, were 0.0076 and 0.061 μg/ml for 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, 0.0153 and 0.122 μg/ml for sinensetin and 0.0305 and 0.122 μg/ml for eupatorin. The percent relative standard deviation (% RSD) values for intraday were 0.048-0.368, 0.025-0.135, and 0.05-0.476 for 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin, respectively, and those for intraday precision were 0.333-1.688, 0.722-1.055, and 0.548-1.819, respectively. The accuracy for intraday were 91.25%-103.38%, 94.32%-109.56%, and 92.85%-109.70% for 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin, respectively, and those for interday accuracy were 97.49%-103.92%, 103.58%-104.57%, and 103.9%-105.33%, respectively. The method was found to be simple, accurate and precise and is recommended for routine quality control analysis of O stamineus extract containing the three flavonoids as the principle components in the extract.
Fulltext Molecular characterization of Leishmania species isolated from cutaneous leishmaniasis in Yemen

Mahdy MA, Al-Mekhlafi HM, Al-Mekhlafi AM, Lim YA, Bin Shuaib NO, Azazy AA, et al.

PLoS One, 2010 Sep 20;5(9).
PMID: 20862227 DOI: 10.1371/journal.pone.0012879

BACKGROUND: Cutaneous leishmaniasis (CL) is a neglected tropical disease endemic in the tropics and subtropics with a global yearly incidence of 1.5 million. Although CL is the most common form of leishmaniasis, which is responsible for 60% of DALYs lost due to tropical-cluster diseases prevalent in Yemen, available information is very limited.
METHODOLOGY/PRINCIPAL FINDINGS: This study was conducted to determine the molecular characterization of Leishmania species isolated from human cutaneous lesions in Yemen. Dermal scrapes were collected and examined for Leishmania amastigotes using the Giemsa staining technique. Amplification of the ribosomal internal transcribed spacer 1(ITS-1) gene was carried out using nested PCR and subsequent sequencing. The sequences from Leishmania isolates were subjected to phylogenetic analysis using the neighbor-joining and maximum parsimony methods. The trees identified Leishmania tropica from 16 isolates which were represented by two sequence types.
CONCLUSIONS/SIGNIFICANCE: The predominance of the anthroponotic species (i.e. L. tropica) indicates the probability of anthroponotic transmission of cutaneous leishmaniasis in Yemen. These findings will help public health authorities to build an effective control strategy taking into consideration person-to-person transmission as the main dynamic of transmission of CL.

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